Insect resistant plants

ABSTRACT

A method for producing genetically transformed plants exhibiting toxicity to Coleopteran insects is disclosed. In another aspect, the present invention embraces chimeric plant genes, genetically transformed cells and differentiated plants which exhibit toxicity to Coleopteran insects. In yet another aspect, the present invention embraces bacterial cells and plant transformation vectors comprising a chimeric plant gene encoding a Coleopteran toxin protein of Bacillus thuringiensis.

This application is a continuation of U.S. application Ser. No.08/435,101, filed May 4, 1995, now abandoned, which is a divisional ofof U.S. application Ser. No. 08/072,281, filed Jun. 4, 1993, now U.S.Pat. No. 5,495,071, which is a continuation of U.S. application Ser. No.07/523,284, filed May 14, 1990, flow abandoned, which is a continuationof U.S. application Ser. No. 07/044,081, filed Apr. 29, 1987, nowabandoned.

BACKGROUND OF THE INVENTION

The present invention relates to the fields of genetic engineering,biochemistry and plant transformation. More particularly, the presentinvention is directed toward transformation of plant cells to express achimeric gene encoding a protein toxic to Coleopteran insects.

Bacillus thuringiensis (B.t.) is a spore forming soil bacterium which isknown for its ability to produce a parasporal crystal protein which istoxic to a wide variety of insects. Most strains are active againstLepidopteran insects (moths and butterflies) and a few are reported tohave activity against Dipteran insects (mosquitoes and flies, seeAronson et al. 1986). Toxin genes from a variety of these strains havebeen cloned and the toxins have been expressed in heterologous hosts(Schnepf et al., 1981; Klier et al., 1982). In recent years, B.t. var.tenebrionis (B.t.t., Krieg et al., 1983; Krieg et al., 1984) and B.t.var. san diego (B.t. sd., Herrnstadt et al., 1986) strains have beenidentified as having activity against Coleopteran insects. The toxingene from B.t.sd. has been cloned, but the toxin produced In E. coli wasreported to be a larger size than the toxin from B.t.sd. crystals, andactivity of this recorabinant B.t.sd. toxin was implied to be weak.

Insects susceptible to the action of the protein toxin ofColeopteran-type Bacillus thuringiensis bacteria include, but are notlimited to, Colorado potato beetle (Leptinotarsa decemlineata), bollweevil (Anthonomus grandis), yellow mealworm (Tenebrio molitor), elmleaf beetle (Pyrrhalta luteola) and Southern corn rootworm (Diabroticaundecimpunctata howardi).

Therefore, the potential for genetically engineered plants which exhibittoxicity or tolerance toward Coleopteran insects was foreseen if suchplants could be transformed to express a Coleopteran-type toxin at ainsecticidally-effective level. Agronomically important crops which areaffected by Coleopteran insects include alfalfa, cotton, maize, potato,rape (canola), rice, tobacco, tomato, sugar beet and sunflower.

Although certain chimeric genes have been expressed in transformed plantcells and plants, such expression is by no means straight forward.Specifically, the expression of Lepidopteran-type B.t. toxin proteinshas been particularly problematic. It has now been found that theteachings of the art with respect to expression of Lepidopteran-typeB.t. toxin protein in plants do not extend to Coleopteran-type B.t.toxin protein. These findings are directly contrary to the priorteachings which suggested that one would employ the same geneticmanipulations to obtain useful expression of such toxins in transformedplants.

SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention, there has beenprovided a method for producing genetically transformed plants whichexhibit toxicity toward Coleopteran insects, comprising the steps of:

(a) inserting into the genome of a plant cell susceptible to attack byColeopteran insects a chimeric gene comprising:

i) a promoter which functions in plant cells to cause production of RNA;

ii) a DNA sequence that causes the production of a RNA sequence encodinga Coleopteran-type toxin protein of Bacillus thuringiensis; and

iii) a 3' non-translated DNA sequence which functions in plant cells tocause the addition of polyadenylate nucleotides to the 3' end of the RNAsequence;

(b) obtaining transformed plant cells, and

(c) regenerating from the transformed plant cells geneticallytransformed plants exhibiting resistance to Coleopteran insects.

In accordance with another aspect of the present invention, there hasbeen provided a chimeric plant gene comprising in sequence:

(a) a promoter which functions in plant cells to cause the production ofRNA;

(b) a DNA sequence that causes the production of a RNA sequence encodinga Coleopteran-type toxin protein of Bacillus thuringiensis; and

(c) a 3' non-translated region which functions in plant cells to causethe addition of polyadenylate nucleotides to the 3' end of the RNAsequence.

There has also been provided, in accordance with another aspect of thepresent invention, bacterial cells, transformed plant cells and planttransformation vectors that contain, respectively, DNA comprised of theabove-mentioned elements (a), (b) and (c).

In accordance with yet another aspect of the present invention, adifferentiated plant has been provided that comprises transformed plantcells, as described above, which exhibit toxicity to Coleopteraninsects. The present invention also contemplates seeds which produce theabove-described transformed plants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the DNA probes used for isolation of the B.t.t. toxin gene.

FIG. 2 shows the steps employed in the preparation of plasmid pMON5432.

FIG. 3 shows the orientation of the 3.0 kb HindIII fragment encoding thetoxin gene in pMON5420 and pMON5421 with respect to the multilinker ofpUC119.

FIG. 4 shows the strategy utilized for sequencing of the B.t.t. toxingene contained in pMON5420 and pMON5421.

FIGS. 5A-5M show the DNA sequence and location of restriction sites forthe 1932 bp ORF of the B.t.t. gene encoding the 644 amino acid toxinprotein.

FIG. 6 shows the bands observed for B.t.t. toxin following SDS-PAGEanalysis.

FIG. 7 shows the N-termini of proteins expressed from the B.t.t. toxingene or proteolytically produced in vivo in B.t.t.

FIGS. 8A-8B represent the altered B.t.t. genes used to analyze thecriticality of the C-terminal portion of the toxin.

FIGS. 9A-9B represent the altered B.t.t. genes used to analyze thecriticality of the N-terminal portion of the toxin.

FIG. 10 shows the deletions produced in evaluation of B.t.t. toxinprotein mutants.

FIG. 11 shows the steps employed in preparation of plasmids pMON9758,pMON9754 and pMON9753.

FIG. 12 shows the steps employed in preparation of plasmid pMON9791.

FIG. 13 shows the steps employed in preparation of plasmid pMON9792.

FIG. 14 shows a plasmid map for plant transformation cassette vectorpMON893.

FIGS. 15A-15B show the steps employed in preparation of plasmidpMON9741.

FIG. 16 shows the steps employed in the preparation of plasmid pMON5436.

FIG. 17 illustrates the elements comprising the T-DNA region of disarmedAgrobacterium ACO.

FIG. 18 shows the DNA sequence for the enhanced CaMV35S promoter.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for transforming plants toexhibit toxicity toward susceptible Coleopteran insects. Moreparticularly, the present invention provides transgenic plants whichexpress the Coleopteran-type toxin protein of Bacillus thuringiensis atan insecticidal level.

In one aspect, the present invention comprises chimeric genes whichfunction in plants and produce transgenic plants which exhibit toxicitytoward susceptible Coleopteran insects. The expression of a plant genewhich exists as double-stranded DNA involves the transcription of onestrand of the DNA by RNA polymerase to produce messenger RNA (mRNA), andprocessing of the mRNA primary transcript inside the nucleus. Thisprocessing involves a 3' non-translated region which adds polyadenylatenucleotides to the 3' end of the mRNA.

Transcription of DNA to produce mRNA is regulated by a region of DNAusually referred to as the "promoter." The promoter region contains asequence of nucleotides which signals RNA polymerase to associate withthe DNA, and initiate the production of a mRNA transcript using the DNAstrand downstream from the promoter as a template to make acorresponding strand of RNA.

A number of promoters which are active in plant cells have beendescribed in the literature. These include the nopaline synthase (NOS),octopine synthase (OCS) and mannopine synthase (MAS) promoters which arecarried on tumor-inducing plasmids of Agrobacterium tumefaciens, thecauliflower mosaic virus (CaMV) 19S and 35S promoters, and thelight-inducible promoter from the small subunit of ribulosebis-phosphate carboxylase (ssRUBISCO, a very abundant plantpolypeptide). These types of promoters have been used to create varioustypes of DNA constructs which have been expressed in plants; see e.g.,PCT publication WO 84/02913 (Rogers et al., Monsanto).

Promoters which are known or are found to cause production of a mRNAtranscript in plant cells can be used in the present invention. Suitablepromoters may include both those which are derived from a gene which isnaturally expressed in plants and synthetic promoter sequences which mayinclude redundant or heterologous enhancer sequences. The promoterselected should be capable of causing sufficient expression to result inthe production of an effective amount of toxin protein to render theplant toxic to Coleopteran insects. Those skilled in the art recognizethat the amount of toxin protein needed to induce the desired toxicitymay vary with the particular Coleopteran insects to be protectedagainst. Accordingly, while the CaMV35S, ssRUBISCO and MAS promoters arepreferred, it should be understood that these promoters may not beoptimal promoters for all embodiments of the present invention.

The mRNA produced by the chimeric gene also contains a 5' non-translatedleader sequence. This sequence may be derived from the particularpromoter selected such as the CaMV35S, ssRUBISCO or M-AS promoters. The5' non-translated region may also be obtained from other suitableeukaryotic genes or a synthetic gene sequence. Those skilled in the artrecognize that the requisite functionality of the 5' non-translatedleader sequence is the enhancement of the binding of the mRNA transcriptto the ribosomes of the plant cell to enhance translation of the mRNA inproduction of the encoded protein.

The chimeric gene also contains a structural coding sequence whichencodes the Coleopteran-type toxin protein of Bacillus thuringiensis oran insecticidally-active Fragment thereof. Exemplary sources of suchstructural coding sequences are B.t. tenebrionis and B.t. san diego.Accordingly, in exemplary embodiments the present invention provides astructural coding sequence from Bacillus thuringiensis var. tenebrionisand insecticidally-active fragments thereof. Those skilled in the artwill recognize that other structural coding sequence substantiallyhomologous to the toxin coding sequence of B.t.t. can be utilizedfollowing the teachings described herein and are, therefore, within thescope of this invention.

The 3' non-translated region contains a polyadenylation signal whichfunctions in plants to cause the addition of polyadenylate nucleotidesto the 3' end of the RNA. Examples of suitable 3' regions are (1) the 3'transcribed, non-translated regions containing the polyadenylate signalof the tumor-inducing (Ti) plasmid genes of Agrobacterium, such as thenopaline synthase (NOS) gene, and (2) plant genes like the soybeanstorage protein genes and the ssRUBISCO. An example of preferred 3'regions are those from the NOS, ssRUBISCO and storage protein genes,described in greater detail in the examples below.

The Coleopteran-type toxin protein genes of the present invention areinserted into the genome of a plant by any suitable method. Suitableplant transformation vectors include those derived from a Ti plasmid ofAgrobacterium tumefaciens such as those described in, e.g. EPOpublication 131,620 (Rogers et al.), Herrera-Estrella 1983, Bevan 1.983,Klee 1985 and EPO publication 120,516 (Schilperoort et al.). In additionto plant transformation vectors derived from the Ti or root-inducing(Ri) plasmids of Agrobacterium, alternative methods can be used toinsert the Coleopteran-type toxin protein genes of this invention intoplant cells. Such methods may involve, for example, liposomes,electroporation, chemicals which increase free DNA uptake, and the useof viruses or pollen as vectors. If desired, more than one gene may beinserted into the chromosomes of a plant, by methods such as repeatingthe transformation and selection cycle more than once.

The plant material thus modified can be assayed, for example, byNorthern blotting, for the presence of Coleopteran-type toxin proteinmRNA. If no toxin protein mRNA (or too low a titer) is detected, thepromoter used in the chimeric gene construct is replaced with another,potentially stronger promoter and the altered construct retested.Alternately, level of toxin protein may be assayed by immunoassay suchas Western blot. In many cases the most sensitive assay for toxinprotein is insect bioassay.

This monitoring can be effected in whole regenerated plants. In anyevent, when adequate production of toxin protein mRNA is achieved, andthe transformed cells (or protoplasts) have been regenerated into wholeplants, the latter are screened for resistance to attack by Coleopteraninsects. Choice of methodology for the regeneration step is notcritical, with suitable protocols being available for hosts fromLeguminosae (alfalfa, soybean, clover, etc.), Unbelliferae (carrot,celery, parsnip), Cruciferae (cabbage, radish, rapeseed, etc.),Cucurbitaceae (melons and cucumber), Gramineae (wheat, rice, corn,etc.), Solanaceae (potato, tobacco, tomato, peppers), Malvaceae (cotton,etc.), Chenopodiaceae (sugar beet, etc.) and various floral crops. Seee.g. Ammirato et al. (1984).

All protein structures represented in the present specification andclaims are shown in conventional format wherein the amino group at theN-terminus appears to the left and the carboxyl group at the C-terminusat the right. Likewise, amino acid nomenclature for the naturallyoccurring amino acids found in protein is as follows: alanine (ala;A),asparagine (Asn;N), aspartic acid (Asp;D), arginine (Arg;R), cysteine(Cys;C), glutamic acid (Glu;E), glutamine (Gln;g), glycine (Gly;G),histidine (His;H), isoleucine (Ile;I), leucine (Leu;L), lysine (Lys;K),methionine (Met;M), phenylalanine (Phe;F), proline (Pro;P), serine(Ser;S), threonine (Thr;T), tryptophan (Trp;W), tyrosine (Tyr;Y) andvaline (Val;V).

Isolation of B.t.t. Toxin Gene

The B.t.t. gene encoding the Coleopterantype toxin protein was isolatedas described below.

Isolation of Protein Crystals

B.t. tenebrionis was grown in Trypticase Soybroth (TSB) medium for theisolation of protein crystals. In attempting to isolate intact crystalsfrom B.t.t. a significant difference between these crystals and those ofthe Lepidopteran-type was noted. While Lepidopteran-type crystals areroutinely isolated on gradients formed from Renografin, Hypaque or NaBr,it was found that B.t.t. crystals dissolved in these gradients media. Itwas found that B.t.t. crystals were stable in gradients of sucrose, andsucrose gradients were used for the isolation of B.t.t.. crystals.

Isolation of B.t.t. Toxin from Crystals

Purified crystals were analyzed for their protein composition by SDSpolyacrylamide gel electrophoresis. Results of these experimentsindicated that B.t.t. crystals contained at least two protein componentswith molecular weights of approximately 68 to 70 kilodaltons (kDa) andapproximately 60 kDa, respectively. The relative amounts of thecomponents were variable from preparation to preparation. In addition,it was suggested that the higher molecular weight component mightconsist of more than a single protein. Bernhard (1986) reported proteinsof about 68 kDa and 50 kDa as components of B.t.t. crystals. Herrnstadtet al. (1986) reported that the crystals of B.t. san diego were composedof a protein of about 64 kDa. In contrast, Lepidopteran-type B.t.strains such as B.t. kurstaki typically contain a higher molecularweight protein of 130 kDa to 140 kDa. This result indicates asignificant difference in the structure of the Lepidopteran andColeopteran toxin proteins.

Several approaches were taken to purifying the individual proteincomponents of the crystal. Isoelectric focusing was not successfulbecause all of the protein precipitated. Anion exchange high pressureliquid chromatograph (HPLC) on a Mono Q column failed to resolve thecomponents. Cation exchange HPLC on a Mono S column in the presence of4M urea resolved five peaks. Analysis of the peaks by SDS gelelectrophoresis indicated that peak A contained only the highermolecular weight band from whole crystals. Peak B was rich in thishigher band with small amounts of the lower band. Peak C was rich in thelower band with significant amounts of the upper band. Peaks D and Ewere mixtures of both bands. In most preparations the higher molecularweight band, corresponding to peaks A and B, was the predominant proteinin the crystals. For the HPLC separated material, peaks A and Brepresented most of the recovered protein.

The N-terminal amino acid sequences corresponding to peaks A, B, and Cwere determined. Peaks A and B were found to have the same N-terminalsequence while the peak C sequence was different. The sequencesdetermined were: ##STR1## Insect Toxicity of B.t.t. Proteins

Several preparations of B.t.t. and B.t.t. proteins were tested fortoxicity to various insects including both Lepidopterans andColeopterans. No activity was observed towards Lepidopterans (cornearworm, black cutworm, tobacco hornworm and cabbage looper). Among theColeopterans, activity was observed against Colorado potato beetle(Leptinotarsa decemlineata) and boll weevil (Anthonomus grandis). Lowerlevel activity was exhibited against Southern corn rootworm (Diabroticaundecimpunctata howardi). Insecticidal activity was found in crudebacterial cultures, purified crystals, solubilized crystals and isolatedpeaks C, D, E (pooled), A and B.

Assays for toxicity to Colorado potato beetle were carried out byapplying the preparation to be tested to tomato leaves and allowing theinsects to feed on the treated leaves for four days. Assays with bollweevil and Southern corn rootworm were performed by incorporating thetest material in an appropriate diet mixture.

Identification and Cloning of the B.t.t. Toxin Gene in E. Coli andPseudomanas

Using this N-terminal protein sequence information, synthetic DNA probes(FIG. 1) were designed which were used in the isolation of clonescontaining the B.t.t. toxin gene. Probes were endlabeled with λ-³² P!ATP according to Maniatis (1982). B. thuringiensis var. tenebrionis wasgrown for 6 hours at 37° C. in Spizizen medium (Spizizen, 1958)supplemented with 0.1% yeast extract and 0.1% glucose (SPY) forisolation of total DNA. Total DNA was isolated from B.t.t. by the methodof Kronstad (1983). Cells were grown on Luria agar plates for isolationof B.t.t. crystals used in toxicity studies.

E. coli and Pseudomonas cultures were routinely grown in Luria Broth(LB) with ampicillin (Ap, 200 μg/ml), kanamycin (Km, 50 μg/ml), orgentamicin (Gm, 15 μg/ml) added for plasmid selection and maintenance.

Isolation and Manipulation of DNA

Plasmid DNA was extracted from E. coli and Pseudomonas cells by themethod of Birnboim and Doly (1979) and large quantities were purifiedusing NACS-52 resin (Bethesda Research Laboratories) according tomanufacturer's instructions. Restriction endonucleases, calf alkalinephosphatase and T4 DNA ligase were used according to manufacturer'sinstructions (New England Biolabs). Restriction digestion products wereanalyzed on 0.8% agarose gels electrophoresed in Tris-acetate buffer.DNA fragments for cloning were purified from agarose using thefreeze-thaw method. Construction of recombinant DNA molecules wasaccording to Maniatis et al. (1982). Transformation into E. coli wereperformed according to Maniatis (1982).

Cloning of the B.t.t. Toxin Gene

Southern analysis (Southern, 1975) was performed using the modifieddried gel procedure (Conner et al., 1983). Colony filter hybridization,for detection of B.t.t. toxin clones, used the tetramethylammoniumchloride method (Wood et al., 1985).

Southern analysis of BamHI and HindIII digested B.t.t. total DNAidentified a 5.8 kb BamHI and a 3.0 kb HindIII fragment which hybridizedto the synthetic Al probe. BamHI fragments of B.t.t. DNA (5.4-6.5 kb)were purified from agarose gels and ligated to alkaline phosphatasetreated BamHI digested pUC119. pUC119 is prepared by isolating the 476bp HgiAI/DraI fragment of bacteriophage M13 and making the ends of thefragment blunt with T4 DNA polymerase (New England Biolabs). Thisfragment is then inserted into pUC19 that has been digested with NdeIand filled with Klenow DNA polymerase (New England Biolabs). The ligatedB.t.t. and pUC119 DNA was then used to transform E. coli JM101 cells.After several attempts only 150 Ap resistant colonies were obtained.HindIII fragments of B.t.t. DNA (2.8-3.5 kb) were also cloned into theHindIII site of pUC119, and 1100 colonies were obtained. All colonieswere screened by colony hybridization to the Al probe (FIG. 1). ElevenHindIII clones showed strong hybridization, but none of the BamHIcolonies showed any hybridization. The colonies identified byhybridization to Al were then screened using synthetic probe A2 (FIG. 1)and two colonies showed hybridization to the second probe. Restrictiondigest patterns of the two colonies indicated that the same 3.0 kbHindIII fragment was contained in both but in opposite orientations.These clones were designated pMON5420 and pMON5421 (FIG. 3). To confirmthat the clones did contain the gene for the B.t.t. toxin protein, thesingle stranded DNA from both clones was sequenced using degenerateprobes A1 and A2 as primers for di-deoxy sequencing (Sanger, 1977).Sequence analysis with A1 probe as primer revealed an open reading frame(ORF) whose sequence was identical to amino acids 9 through 15 of theamino acid sequence determined for purified peaks A and B of the B.t.t.toxin protein. Probe A2 produced DNA sequence which began beyond the endof the determined amino sequence, but this DNA sequence was identical tosequence produced with A1. These results confirm that the desired B.t.t.toxin gene was cloned.

Southern hybridization to total B.t.t. DNA using degenerate probes basedon the N-terminus of peak C failed to detect specific bands suggestingthat the amino acid sequence determined for peak C was incorrect or mostprobably was obtained from a mixture of two or more proteins comprisingpeak C.

Analysis of Proteins Produced in E. coli

B.t.t. crystal proteins and recombinant B.t.t. proteins were examined bySDS-PAGE (Laemmli, 1970). One ml of E. coli was centrifuged, the pelletsresuspended in 100 μg SDS-sample buffer and 10 μl samples wereelectrophoresed on 7.5% polyacrylamide gels. The gels were eitherstained with Coomassie Blue or probed for cross reactivity to antibodiesraised against purified B.t.t. toxin crystals. Western Blots wereperformed using the horseradish peroxidase conjugated antibody procedure(Towbin et al., 1984). High molecular weight markers were purchased fromBioRad.

Further confirmation that the clones produced B.t.t. toxin was obtainedby Western blot analysis of the proteins produced in E. coli. E. coliJM101 cells containing either pUC119, pMON5420 or pMON5421 were grownovernight in the presence of IPTG (0.1 μM) to induce the lac promoter.Duplicate samples were analyzed by SDS-PAGE along with purified B.t.t.crystal proteins included as controls. Western blot analysis of one gelrevealed the production of 2 cross reacting proteins by E. colicontaining pMON5420 or pMON5421. These proteins were identical in sizeto the major and minor proteins of the B.t.t. crystal. Molecular weightsof the proteins were determined by comparison to the molecular weightstandards on the second gel stained with Coomassie blue. The major toxinprotein was determined to be 74 kDa in size and the minor toxin proteinwas determined to be 68 kDa in size. The level of B.t.t. toxin proteinsproduced by pMON5420 was increased by the addition of IPTG whileproduction of toxin proteins by pMON5421 was unaffected.

Production of B.t.t. Toxin(s) in Pseudomonas fluorescens

A broad host range vector, pMON5432, was constructed by cloning BamHIdigested pMON5420 into the BamHI site of pMON7111 as shown in FIG. 2.This vector was then mated into P. fluorescens 701El for analysis oftoxin production. Tri-parental matings into Pseudomonas fluorescens weredone as previously described (Ditta et al., 1980). Samples of overnightcultures, grown with and without IPTG, were prepared for Western blotanalysis and insect toxicity studies. The proteins produced byPseudomonas were identical in size to the E. coli produced proteins andprotein expression was increased with the addition of IPTG.

Insect Toxicity Assay

Coleopteran toxin activity was assayed using newly hatched Coloradopotato beetle (Leptinotarsa decemlineata) insects in a tomato leaffeeding assay. E. coli and Pseudomonas cultures were grown overnight inthe presence of IPTG, centrifuged and resuspended at variousconcentrations in 10 mM MgSO₄. The cells were disrupted by sonication(three 15 sec. pulsed treatments on ice). Tween-20 (0.1%) was added andthe sample painted onto a tomato leaf placed into a 9 cm petri dishlined with moist filter paper. Ten Colorado potato beetle larvae wereadded to each leaf. After four days, the percentage corrected mortality(percentage of insects alive in the control minus the percentage ofinsects alive in the treated sample divided by the percentage alive inthe control) was computed using Abbott's formula (Abbott, 1925). Assayswere performed in duplicate and the data combined. B.t.t. crystal/sporepreparation were used as positive controls.

E. coli cultures of pMON5420 and pnMON5421 were evaluated forColeopteran toxicity using different concentrations of cultures grownwith added IPTG. A comparison of recombinant and wild type B.t.t. toxinactivities is shown below in Table a. The results show that therecombinant B.t.t. protein(s) are toxic to Colorado potato beetle. The2×-concentrated, IPTG-induced pMON5420 culture killed 100% of theinsects as did the B.t.t. spore/crystal control. These toxicity resultsdemonstrate that the B.t.t. gene cloned was the gene that encodes theB.t.t. toxin protein.

Insect feeding assay showed that the Pseudomonas produced toxins weretoxic to Colorado potato beetle. The relative toxicity of Pseudomonascultures was consistent with the amount of toxin protein produced asdetermined by Western blot analysis when compared to E. coli cultures.

                  TABLE I                                                         ______________________________________                                        Coleopteran Toxicity of Recombinant B. t. t. Toxin                                                        Corrected                                         Sample.sup.1     Concentration.sup.2                                                                      Mortality                                         ______________________________________                                        E. coli JM101                                                                 pUC119           2×    0%                                               pMON5420         1×   83%                                               pMON5420         2×   100%                                              pMON5421         1×   44%                                               pMON5421         2×   61%                                               P. fluorescens 701E1                                                          pMON5432         3×   60%                                               B. t. t. prep               100%                                              ______________________________________                                         .sup.1 Cultures were grown overnight with added IPTG, concentrated,           sonicated and tested for toxicity.                                            .sup.2 1× equals cellular concentration of overnight culture.      

Sequence of Toxin Gene of B.t.t.

Location and orientation of the B.t.t. gene within the cloned fragmentwas determined based on the following information: a) DNA sequence wasobtained from the single stranded pMON5421 template, b) A PstI siteidentified, by DNA sequence analysis, near the start of translation wasmapped in pMON5420 and pMON5421, c) several other restriction sites weremapped, d) a deletion from a BglII site to a BamHI site which deletes130 bp was constructed and both full-length proteins were produced. Thisinformation was used to construct maps of pMON5420 and pMON5421.Referring to FIG. 4, the toxin coding region begins 500 bp from the 5'HindIII site, and 150 bp upstream of the PstI site. The coding regionends approximately 450 bp from the 3' HindIII site. The BglII site isapproximately 350 bp downstream of the stop codon.

Plasmids

The plasmids generated for sequencing the B.t.t. insecticidal toxin geneare listed in Table II. The parental plasmids, pMON5420 and pMON5421,are independent isolates of the HindIII fragment cloned into pUC119 inopposite orientation.

                  TABLE II                                                        ______________________________________                                        Sequencing Plasmids                                                           ______________________________________                                        pMON5420     3.0 HindIII insert from B.t.t. DNA                                            (parent plasmid)                                                 pMON5421     3.0 HindIII insert from B.t.t. DNA                                            (parent plasmid)                                                 pMON5307     EcoRI deletion of pMON5420                                       pMON5308     EcoRI deletion of pMON5421                                       pMON5309     PstI deletion of pMON5420                                        pMON5310     XbaI deletion of pMON5421                                        pMON5311     EcoRV-SmaI deletion of pMON5421                                  pMON5312     NdeI-BamHI deletion of pMON5421*                                 pMON5313     NdeI-BamHI deletion of pMON5420*                                 pMON5314     AsuII-BamHI deletion of pMON5421*                                pMON5315     AsuII(partial)-BamHI deletion of                                              pMON5421*                                                        pMON5316     AsuII-BamHI deletion of pMON5421**                               pMON5426     BglII-BamHI deletion of pMON5420                                 pMON5427     EcoRV-SmaI deletion of pMON5420                                  pMON5428     HpaI-SmaI deletion of pMON5420                                   pMON5429     XbaI deletion of pMON5420                                        ______________________________________                                         *  After digestion of the DNA with both enzymes, the ends were filled in      with Klenow polymerase, ligated and used to transform JM101.                  **  Generation of the AsuIIBamHI deletion of this construct resulted in a     rearrangement of an AsuII fragment to an orientation opposite to its          original location. This resulted in a sequence of 5316 reading toward the     NH.sub.2 end.                                                            

Preparation of Single Stranded Template for Sequencing

The following protocol provides reproducibly good yields of singlestranded template for sequencing. A single colony containing the pUC119with the fragment to be sequenced was streaked on L-agar (10 g tryptone,5 g yeast extract, 5 g Nacl, and 15 g agar per liter) containingampicillin (200 μg per ml). A single colony from this plate wasinoculated into 3 ml of L-broth (200 μg per ml ampicillin) and incubatedat 37° C. overnight with shaking. From this culture, 50 μl wasinoculated into 10 ml of 2×YT (20 g tryptone and 10 g yeast extract perliter) with 200 μg of ampicillin per ml in a 150 ml side arm flask andincubated at 37° C. with shaking. After 2-3 hours (Klett reading of 50),100 μl of M13K07 (helper phage) grown in E. coli JM101 was added toinduce the culture. The flask was shaken for one hour followed by theaddition of 20 ml of 2×YT adjusting the final concentration of kanamycinto 70μg per ml and ampicillin to 200 μg per ml. The cultures were shakenfor 16-18 hours at 37° C. A total of three mls of the induced overnightculture was found to be sufficient to isolate a suitable amount oftemplate for four sequencing experiments. The three mls were spun in 1.5ml eppendorf tubes for 1 minute, decanted and filtered through a 0.2 μmGelman Sciences Acrodisc®. This step was found to be useful for theremoval of cellular debris and intact E. coli. A polyethylene glycolprecipitation (20% PEG, 2.5M NaCl, 500 μl per 2 ml of lysate) at roomtemperature for 10 minutes was followed by centrifugation for 10minutes. The supernatant was discarded followed by a brief spin (15seconds) and removal of the residual PEG. Any remaining PEG will becarried through the template isolation and adversely affect DNAsequencing reactions. The pellets are resuspended in 100 μl of TE (10 mMTris, 1 mM EDTA, pH 8.0), combined and mixed well with 200 μl ofbuffered phenol (buffered by equilibration with an equal volume of 1MTris-HCl, pH 8.0, then 0.1M Tris-HCl, pH 8.0, followed by an equalvolume of TE). After incubation at 55° C. for 10 minutes an equal volume(200 μl) of phenol/chloroform (1::1) was added, vortexed, andcentrifuged for 2 minutes. The top layer was removed, extracted with 200μl of chloroform, centrifuged and the aqueous phase removed. The singlestranded template was precipitated with 25 μl of 3M sodium acetate (pH5.2) and 600 μl of 95% ethanol, incubated on dry ice for 5 minutes andcentrifuged for 10 minutes. The precipitate was resuspended in 25 μl ofH₂ O and 2 μl was checked on an agarose gel for correct size, relativeconcentration and contaminating DNA.

Sequencing Reagents and Conditions

The protocols for DNA sequencing are described in detail in the Handbookavailable from Amersham Corporation. Reagents (nucleotides, primer,buffer, chase solution and Klenow polymerase) were obtained from theAmersham M13 sequencing kit (catalog #N4502). The sequencing mixesprovided in the Amersham kit were adjusted for efficient sequencing ofthe A-T rich B.t.t. gene. Instead of the recommended 1::1 mix of dNTP toddNTP, the following ratios were found to be more appropriate; 40 μldATP: 10 μl ddATP, 35 μl dTTP: 15 μl ddTTD, 15 μl dGTP: 35 μl ddGTP, and10 μl dCTP: 40 μl ddCTP. Radioactive sulfur ( α-³⁵ S! dATP) was used inthe sequencing reactions (Amersham catalog #SJ.1304). The sequencinggels (prepared as described in the Amersham handbook) were run on theHoeffer "Poker Face" apparatus at 70 watts (1200-1400 volts) which wasfound to give very good resolution. Higher voltages resulted in fuzzybands.

Sequencing of the B.t.t. Toxin Gene

The isolated plasmids, pMON5420 and pMON5421, contained a 3.0 HindIIIfragment in opposite orientation (see FIG. 3). The major protein of theB.t.t. crystal, which was used as the basis for design of theoligonucleotide probes, has a molecular weight estimated to be 73-76kdal corresponding to approximately 2.0 kb of DNA. Initial sequencingfrom the A1 and A2 primers (synthetic oligonucleotides based on theamino acid sequence of Peak A; see Table III, below) confirmed that theDNA sequence corresponded to the anticipated amino acid sequence.

                  TABLE III                                                       ______________________________________                                        Synthetic Oligonucleotides Used for Sequencing                                the B. t. t. Insecticidal Toxin Gene                                          Primer  Template   Sequence       Location.sup.1                              ______________________________________                                        Bttstart                                                                              pMON5420   tgaacatggttagttgg                                                                            291-275                                     Bttext  pMON5421   taggtgatctctaggcg                                                                            422-439                                     Bttseq  pMON5421   ggaacaaccttctctaatat                                                                         1156-1175                                   BttA1*  pMON5421   atgaayccnaayaaycg                                                                            205-222                                     BttA2*  pMON5421   garcaygayacyathaa                                                                            227-242                                     ______________________________________                                         y = t or c.  r = a or g.  h = t, c or a.  n = a, g, c or t.                   .sup.1 The location of the primers is based on the total of 2615 bases        sequenced. Sequencing from pMON5420 proceeded toward the amino acid end       and from pMON5421 toward the carboxyl end (see Fig. 3).                  

A PstI site was located in the initial sequence which was used toidentify the location and probable orientation of the B.t.t. gene withinpMON5420 and pMON5421 (see FIGS. 3 and 4). Mapping of restriction siteswith a number of enzymes (HpaI, XbaI, NdeI, EcoRV, and BglII) and thenumerous unique sites remaining in the pUC119 portion of both pMON5420and pMON5421 provided the opportunity to obtain sequence using theuniversal sequencing primer. Deletions were generated in both pMON5420and pMON5421 bringing the universal primer homologous region in closeproximity to internal regions of the gene. In areas not easily sequencedby generating deletions, synthetic oligonucleotides corresponding tosequenced regions in the coding sequence (Table III) were used asprimers to obtain extensions of the sequenced regions. The regionssequenced (sequence coordinates; Table IV) and the direction ofsequencing is depicted in FIG. 4.

                  TABLE IV                                                        ______________________________________                                        Source of Sequence Data                                                                      Length                                                         Plasmid        (bp)    Location                                               ______________________________________                                        pMON5307       414      797-1211                                              pMON5308       276     1895-2171                                              pMON5309       170     114-284                                                pMON5310       283     1595-1880                                              pMON5311       110     1812-1922                                              pMON5312       248      782-1030                                              pMON5314       291     2041-2305                                              pMON5315       330     1157-1187                                              pMON5316       153     1861-2041                                              pMON5426       300     2220-2520                                              pMON5427       110     1701-1812                                              pMON5428       129     1548-1677                                              pMON5429       303     1292-1595                                              Bttstart       264      1-264                                                 Bttext         380     440-820                                                BttA2          267     250-517                                                ______________________________________                                    

Computer Analysis of the B.t.t. Insecticidal Toxin Gene

A total of 2615 base pairs of sequence were obtained from pMON5420 andpMON5421. Computer analysis of the sequence revealed a single openreading frame from base pair 205 to 2136. Referring to FIG. 5, theB.t.t. insecticidal toxin gene is 1932 base pairs, coding for protein of644 amino acids with a molecular weight of 73,091 daltons. The proteinhas a net charge of -17 and a G-C content of 34%.

Comparison Between Coleopteran-type and Lepidopteran-type Toxin Genesand Proteins

Although the Coleopteran-type toxins and the Lepidopteran-type toxinsare derived from Bacillus thuringiensis, there are significantdifferences between the toxin genes and the toxin proteins of the twotypes. As isolated from Bacillus thuringiensis both types of toxins arefound in parasporal crystals; however, as described above, thesolubility properties of the crystals are distinctly different. Inaddition, the sizes of the toxin proteins found in solubilized crystalsare completely different. Lepidopteran-type toxin proteins are typicallyon the order of 130 kDa while the Coleopteran-type toxin proteins areapproximately 70 kDa.

Isolation and DNA sequence analysis of the Coleopteran-type toxin genefrom B.t. tenebrionis predicts the amino acid sequence of the toxinprotein (see FIG. 5). Both the nucleotide sequence and the derived aminoacid sequence of the Coleopteran-type toxin gene have been compared tonucleotide and amino acid sequence of a typical Lepidopteran-type toxin.This comparison was performed using the computer program BESTFIT ofDevereux et al (1984) which employs the algorithm of Smith and Waterman(1981). BESTFIT obtains maximum alignment of two nucleotide or aminoacid sequences. BESTFIT calculates two parameters, quality and ratio,which can be used as alignment metrics when comparing differentalignments. Ratio varies between 0 and 1.0. A larger ratio indicates abetter alignment (greater similarity) between two sequences.

The BESTFIT alignment shows that the two types of toxin genes arerelated at both the nucleotide sequence and amino acid sequence level.However, the alignment also shows that the two sequences are clearlydistinct and possess many regions of mismatch at: both the nucleotideand amino acid sequence levels. For example, the ratio for comparison ofthe two amino acid sequences is only 0.22. At the nucleotide sequencelevel, maximum alignment is obtained only by the introduction of manygaps in both sequences, and the ratio is only 0.072.

There are many sequenced examples of Leptidopteran-type toxin genes;similar comparison among these genes has shown that the gene from B.t.kurstaki HD-1 described by Schnepf et al. (1985) and that from B.t.kurstaki HD-73 described by Adang et al. (1985) represent the two mostdivergent Lepidopteran-type toxin genes. By comparison with the ratioscalculated above for alignment of the Colepteran-type and theLepidopteran-type gene, the ratio for amino acid sequence comparison ofthe two most divergent Lepidopteran-type proteins is 0.811, and theratio for these two Lepidopteran-type genes at the nucleotide sequencelevel is 0.755. This indicates that although the Coleopteran-type andLepidopteran-type toxin genes may be evolutionarily related, they arequite distinct in both nucleotide and amino acid sequence.

High Level Production of Recombinant B.t.t. Toxin in E. Coli

To facilitate purification of large quantities of recombinant B.t.t.toxin, it was necessary to clone the B.t.t. gene into an E. coli highexpression vectors. Site directed mutagenesis was used to introduce anNcoI restriction site into pMON5420 at the ATG codon at the start of theopen reading frame.

Site Directed Mutagenesis

Site-directed mutagenesis to introduce new restriction sites wasperformed by the method of Kunkel (1985). Plasmid pMON5420 wasintroduced by transformation into E. coli strain BW313, which containsthe dut⁻ and ung⁻ mutations in order to incorporate deoxyuridine intothe DNA. A single transformed colony was grown overnight in 2×YT mediumcontaining 100 μg/ml ampicillin and 0.25 μg/ml uridine. A 0.5 ml aliquotof this culture was added to 10 al of the same medium and incubated forone hour at 37° C. with vigorous shaking to a density of 0.23 (A600). Toinduce formation of single strand containing phage particles, helperphage M13K07 was added at a multiplicity of approximately 10 andincubation was continued for one hour to a density of 0.4 (A600). Theculture was diluted by addition of 30 ml of the above medium, andkanamycin was added to a final concentration of 70 μg/ml. Incubation wascontinued for 15 hours at which point cells were removed bycentrifugation. Phage particles were precipitated from 25 ml ofsupernatant by addition of 5 ml of 20% PEG/2.5M NaCl/50 μg/ml RNAase Afollowed by incubation on ice for 15 minutes. Phage were recovered bycentrifugation and dissolved in 0.8 ml TE buffer. DNA was isolated fromthe particles by three extractions with 0.8 ml phenol/chloroform/isoamylalcohol (25:24:1) followed by ethanol precipitation. The DNA pellet wasdissolved in 100 μl of water to a final concentration of approximately 1mg/ml (estimated by agarose gel electrophoresis).

Synthetic oligonucleotide primers for mutagenesis were suspended inwater at a concentration of approximately 10 pmole/μl. Theoligonucleotides were phosphorylated utilizing T4 polynucleotide kinasein a reaction containing 50 pmoles oligonucleotide, 1 mM ATP, 25 mMTris-HCl pH 8, 10 MM MgCl₂, 0.2 mM spermidine-HCl, 1 mM DTT and 2 unitsof enzyme. The reaction was incubated at 37° C. for 30 minutes and thenheated at 70° C. for 5 minutes. The phosphorylated primer was annealedto the deoxyuridine containing phage DNA by mixing approximately 1 pmoleof the phage DNA (2 μg) with 10 pmole primer in a reaction containing6.6 mM Tris-HCl, 6.6 EM MgCl₂, 6.6 mM NaCl and 5 mM DTT. The mixture washeated to 70° C. for seven minutes and then slowly cooled to roomtemperature. The annealed primer/template was used as the substrate forsynthesis of double-stranded, closed circular DNA by addition of eachdNTP to 0.5 mM, ATP to 0.5 mM, 5 units of Klenow fragment DNA polymeraseand 400 units T4 DNA ligase (New England Biolabs). The reaction wascarried out in the same buffer salts as for annealing at 15° C. forapproximately 15 hours. At this time an additional 400 units of ligasewas added and incubation was continued for two hours.

One half of the reaction was used to transform 0.15 ml of CaCl₂ -treatedJM101 cells, and the cells were spread on LB plates containing 100 μg/mlampicillin. Between 30 and several hundred colonies were recovered foreach mutagenesis reaction. Single colonies were grown overnight in LBcontaining ampicillin and plasmid minipreps were prepared by thealkaline SDS method. Plasmids were analyzed for the presence of the newrestriction site and the presence of the site was confirmed by sequenceanalysis as described above.

A plasmid containing a NcoI site (pMON9759) at the start of the B.t.t.insecticidal toxin gene was generated by site-specific mutagenesis. Theprimer used is shown below: ##STR2## The generation of the NcoI site atthe N-terminus has changed the second amino acid from asparagine toaspartic acid. This change does not affect insect toxicity. BamHI andStyI sites have also been generated as a consequence of the introductionof this NcoI site. The plasmid containing the NcoI site has beendesignated pMON9759. The 2.5 kb NcoI-HindIIl fragment containing thetoxin encoding segment from pMON9759 was then cloned into NcoI-HindIIIdigested. pMON5634 to produce pMON5436. Referring to FIG. 16, pMON5634is a pBR327 based plasmid which also contains the f1 phage origin ofreplication. The vector contains a synthetic recA promoter which isinduced by nalidixic acid. The gene 10 leader from phage T7 (describedin commonly assigned pending U.S. Pat. application Ser. No. 005821,filed Feb. 4, 1987, the disclosure of which is hereby incorporated byreference) is also present to increase expression in E. coli. Asynthetic linker with multiple cloning sites was added for insertion ofgenes downstream of the promoter and gene 10 leader sequence.

For induction of the recA promoter, overnight cultures were diluted 1:50into M9 minimal media (Miller, 1972) with 0.2% casamino acids and 0.25%glucose added. At 150 Klett units, naladixic acid was added to 50 μg/mland cells were harvested 3 hours post induction. The level of B.t.t.toxin produced by nalidixic acid induced pMON5436 was compared to IPTGinduced pMON5420 by analysis on SDS-PAGE. The Coomassie blue stained gelrevealed no detectable B.t.t. produced by pMON5420 while the level ofB.t.t. produced by pMON5436 was approximately 5% of total protein. Thisconstruct was used to isolate large quantities of the recombinant B.t.t.toxin proteins to investigate toxicity levels, insect specificity, andmode of action.

B.t.t. Toxin Characterization

Identification of the Number and Origin of the B.t.t. Proteins

B.t. var. tenebrionis produces a number of Coleopteran-type toxinproteins, present in protein crystals, which are producedco-incidentally with sporulation (see FIG. 6). These protein crystalsare released into the media as cells autolyse during or followingsporulation. To determine the number of toxin proteins produced by B.t.var. tenebrionis, 500 ml cultures of this organism were grown in 2 literflasks in 15% TSB medium in 100 mM 2-(N-morpholino) ethanesulfonic acid(MES) buffer, pH 7.0 at 30° C. for 7 days. At this point the cultureshave sporulated and the cells lysed. Protein crystals and spores wereharvested by centrifugation at 20,000×gravity (g) for 20 min. at 4° C.Pellets were washed three times with excess water, followed by threewashes with 2 M NaCl. The resultant pellet was stored at 4° C. in waterplus 0.02% sodium azide. B.t.t. toxin protein was solubilized from thecrystals by suspending the pellet in 100 mM sodium carbonate buffer, pH10 and stirring this suspension for two hours at room temperature. Aftercentrifugation 20,000×g for 20 min to remove unsolubilized materials,the supernatant was filtered through a 0.2 μm filter to remove anyremaining spores. B.t.t. toxin protein prepared in this manner, as docrystals solubilized in 125 mM Tris-HCl, 4% SDS, 20% glycerol and 10%2-mercaptoethanol, pH 6.8, (SDS sample buffer used to prepare samplesfor SDS-PAGE analysis) is comprised of four major and different proteinsas judged by SDS-PAGE analysis. Five unique Products were identified byN-terminal amino acid analysis. To determine whether all five of theseproteins were derived from the same gene or whether two or more genesare required for their synthesis, the N-.terminal amino acid sequence ofeach of these proteins were determined using automatic Edman degradationchemistry.

An Applied Biosystems, Inc. Model 470A gas phase sequencer (Foster City,Calif.) was employed (Hunkapiller, et al., 1983). The respectivePTH-amino acid derivatives were identified by RP-HPLC analysis in anon-line fashion employing an Applied Biosystems, Inc. Model 120A PTHanalysis fitted with a Brownlee 2.1 mm I.D. PTH-C18 column.Determination of the N-terminal amino acid sequence of each protein willestablish whether all these proteins were derived from the B.t.t. toxingene described above.

The strategy to sequence these proteins was to sequence the B.t.t. toxinproteins corresponding to bands 1 and 3 (see FIG. 6) from the E. coliclone JM101 (pMON5436), bands 2, 3 and 4 by electro-elution of theproteins produced by B.t. var. tenebrionis from SDS-PAGE gels. Thesequence of B.t.t. 1 and 3 was determined with proteins purified fromJM101 (pMON5436). JM101 (pMON5436), as well as the other E. coliconstructs (pMON5450, 5456 and 5460, infra) produces the B.t.t. in theform of insoluble refractile bodies after cultures are induced for highlevel expression. The E. coli constructs were grown in modified M9 mediaat 37° C. A culture grown overnight was used to inoculate 400 ml of themodified M9 media in 2.4 1 fernbach flasks to an initial startingdensity of 10 Klett units. Nalidixic acid, in 0.1N NaOH, was added tothe cultures at 100 Klett units to a final concentration of 50 μg/ml, toinduce B.t.t. toxin protein expression. After an additional 4 hours ofincubation, cultures were harvested by centrifugation at 20,000×g for 20min. at 4° C. Cell pellets were suspended in water to a densityequivalent to 5000 Klett units per ml and sonicated in an ice bath witha Heat Systems Ultrasonics sonicator at a power of 9, 50% duty cycle fora total of 5 min. The sonicated preparation was centrifuged for 20 min.at 20,000×g at 4° C. Pellets, containing retractile bodies and celldebris, were washed twice with cold water and suspended at 10,000 Klettunit equivalents per ml in water plus 25% sulfolane. After stirring atroom temperature for 2 hours, the solubilized refractile bodypreparations were centrifuged again at 20,000×g at 4° C. to removeunsolubilized materials. Tris-HCl was added to the supernatant to afinal concentration of 50 mM, pH 7.6. The B.t.t. bands 1 and 3 wereco-purified on an HR5/5 MonoQ ion exchange column using a 75 to 200 mMNacl gradient in 50 mM Tris-HCl, 25% sulfolane, pH 7.6. Fractionscontaining B.t.t. bands 1 and 3 were identified by 9% SDS-PAGE analysis,pooled, dialyzed into 100 mM sodium carbonate, pH 10 buffer andconcentrated in Amicon centricon concentrators. B.t.t. toxin proteincorresponding to band 3 was purified from JM101 (pMON5456) in ananalogous manner.

Bands corresponding to 2 alone and bands 3,3' and 4 (see FIG. 6)combined were electroeluted from 7% SDS-PAGE slab gels which were runwith 48 μg of B.t.t. crystals solubilized in 100 mM sodium carbonate, 20mM dithiotheitol (DTT), pH 10 buffer. Gels were stained for 10 min inCoomassie blue R250 and destained in 50% methanol, 10% acidic acid for20 min. Appropriate bands were excised with a razor blade and the B.t.t.protein electro-eluted. Knowing the amino acid sequence, deduced fromthe DNA sequence of the B.t.t. toxin gene cloned in E. coli, all fiveN-termini of these unique proteins were identified (FIG. 7).

Proteins corresponding to band 1 and 3 originated from two independenttranslational initiation events which start at the methionine atpositions 1 and 48 (FIGS. 6 and 7), respectively. Proteins correspondingto B.t.t. bands 2, 3' and 4, observed only in 3t. var. tenebrionis andnot in the E. coli constructs, apparently arise from proteolyticcleavage of either bands 1 or 3. These results establish that all fiveproteins originate from the same gene.

Purification of B.t.t. Bands 1 and 3 for Insect Toxicity Testing

The B.t.t. proteins produced in E. coli corresponding to bands 3 and 1plus 3 which were solubilized in 25% sulfolane and purified by MonoQchromatography for N-terminal amino acid sequence analysis showed noinsect toxicity against Colorado potato beetle insects. In subsequentexperiments, it was demonstrated that sulfolane itself inactivatesB.t.t. Therefore, an alternative purification method was developed andused compare the relative insecticidal toxicities of B.t.t. bands 1 and3 produced in E. coli compared to the B.t.t. solubilized from nativecrystals of B.t. var. tenebrionis. Cultures were grown, induced,harvested and refractile bodies isolated as described above. The variousB.t.t. proteins were solubilized from the refractile bodies using 100 mMsodium carbonate, pH 10. The solubilized B.t.t. toxin, concentratedusing Amicon stirred cells with YM-10 membranes, was purified on aPharmacia Superose12, gel filtration FPLC column., which separatesB.t.t. bands 1 and 3 and from other contaminating proteins. Appropriatefractions, based upon SDS-PAGE analysis, were pooled, concentrated andused for insect toxicity experiments with the Colorado potato beetleinsects. Proteins corresponding to band 1 (pMON5436, band 1 (pMON5460)and band 3 (pMON5456) were greater than 90% pure based upon SDS-PAGEanalysis. Band 1 produced by pMON5460 has isoleucine at amino acid 48 inplace of methionine (see below).

To obtain native protein toxin from B.t. var. tenebrionis for toxicitycomparisons, native crystals were isolated and purified using sucrosegradient centrifugation as described above. Crystals were solubilized in100 mM sodium carbonate, 20 mM DTT, pH 10 and used for insect toxicitytests.

All B.t.t. toxin protein preparations and controls for insect assaycontained 0.3% Tween 20, a surfactant which enhances the ability ofthese solutions to bind to tomato leaves. Insect toxicity experimentswere performed by thoroughly painting the upper and lower surfaces of 3to 4 week old detached tomato leaves with buffer solutions containingthe designated B.t.t. proteins at the indicated protein concentrations.After the solutions were air dried on the surface of the tomato leaves,a single leaf and 10 Colorado potato beetle insects were placed in apetri dish and incubated at 22° C. for 4 days. The number of deadinsects was determined and the toxicity results expressed as % correctedmortality (%CM); according to Abbott's formula described above. Allexperiments were performed in duplicate and all but the B.t.t. band 1from pMON5460 were repeated on different days. The results of thesetests are shown in the table below.

                  TABLE V                                                         ______________________________________                                        Toxicity of B. t. t. Proteins                                                 Against Colorado Potato Beetle                                                               Concentration                                                                            Corrected                                           Sample         (ug/ml)    Mortality (%)                                       ______________________________________                                        B. t. t. Solubilized                                                                         100        100                                                                20         70                                                                 4          10                                                  Purified Band 1                                                                              100        87                                                  (pMON5436)     20         68                                                                 10         34                                                  Purified Band 1                                                                              100        67                                                  (pMON5460)     20         72                                                                 10         44                                                  Purified Band 3                                                                              100        91                                                  (pMON5456)     20         64                                                                 10         32                                                  ______________________________________                                         Relative toxicity of purified proteins from different E. coli constructs      were compared to solubilized native B. t. t. crystals. Band 1 (pMON5436)      and Band 3 (pMON5456) were purified as described. Band 1 (pMON5460) was       purified using gel filtration chromatography. Native B. t. t. crystals        were solubilized in 100 mM Na.sub.2 CO.sub.3, pH 10.                     

The amounts of B.t.t. toxin required to kill 50% of the Colorado patatobeetle insects were essentially identical for B.t.t. band 1 isolatedfrom pMON5436 and pMON5460 and B.t.t. band 3 isolated from pMON5456(Table V). Likewise, all of these purified B.t.t. preparation from E.coli demonstrated toxicities essentially identical to that observed withthe sodium carbonate solubilized native toxin from B.t. var.tenebrionis.

Determination of Toxic Fragments of B.t.t. Toxin Proteins

Several groups (Schnepf et al. 1985, Hofte et: al. 1986, and Wabiko etal. 1986) have reported that C-terminal truncations of theLepidopteran-type toxins do not reduce toxicity (of the 1155 amino acidsa truncation to amino acid 607 did not result in a loss of toxicity).Therefore, the C-terminal half of the protein is not required fortoxicity. Others have also reported that the Lepidopteran-type toxingenes which contain C-terminal deletions are more highly expressed intransformed plants. There are also reports that to retain toxicity, onlysmall truncations can be made at the N-terminus (Schnepf et al. 1985,and Hofte et al. 1986). Contrary to those teachings it has now beenfound that the Coleopterantype toxin of B.t.t. has substantiallydifferent properties. That is, the C-terminal portion appears to becritical for toxicity therefore permitting essentially no truncations.However, N-terminal deletions can be made and maintain toxicity. Thesedifferences were uncovered using the constructs described below:

Construction of pMON5426 (BglII/BamHI Deletion)

pMON5420 was digested with BglII and BamHI, ligated and transformed intoJM101 to create pMON5426. This deletion was constructed to confirm thatthe BglII site was not within the coding region of the B.t.t. toxingene.

Construction of pMON5438 (HpaI, C-terminal Deletion of 463 be)

pMON5420 was digested with HpaI and ligated with the following syntheticterminator linker. The linker contains nonsense codons in each readingframe and a BglII 5' overhang.

5'-TAGTAGGTAGCTAGCCA-3'

3'-ATCATCCATCGATCGGTCTAG-5'

The ligation was digested with BglII, to remove multiple linker insertsand then re-ligated. The ligation was transformed into JM101 andpMON5430 was isolated. To generate a NcoI site at the start of thetruncated gene, the 2.32 kb PstI fragment of pMON9759 was replaced withthe 1.47 kb PstI fragment of pMON5430 and the new construct wasdesignated pMON5434. The 1.57 kb NcoI/HindIII fragment from pMON5434 wascloned into the E. coli high expression vector pMON5634, to createpMON5438.

Construction of pMON5441 (EcoRV, C-terminal Deletion of 327 bp)

pMON5420 was digested with EcoRV and ligated with the syntheticterminator linker. The ligation was digested with BglII, to removemultiple linker inserts and then re-ligated. The ligation wastransformed in JM101 and pMON5431 was isolated. To generate a NcoI siteat the start of the truncated gene, the 2.32 kb PstI fragment ofpMON9759 was replaced with the 1.61 kb Pst fragment of pMON5431, and thenew construct was designated pMON5435. The 1.71 kb NcoI/HindIII fragmentfrom pMON5435 was cloned into the E. coli high expression vectorpMON5433 to create pMON5441.

Construction of pMON5449 (Bal3l, C-terminal Deletion of 190 bp)

BglII digested pMON9759 was treated with Bal31 nuclease for 5 min.following the manufacturer's instructions. The DNA was electrophoresedin a 0.8% agarose gel and purified from the agarose by the freeze thawmethod. The synthetic terminator linker was then ligated to the purifiedDNA and pMON5442 was isolated. The NcoI/BglII fragment of pMON9759 wasreplaced with the truncated gene fragment from pMON5442 to createpMON5445. The NcoI/HindIII fragment from pMON5445 was cloned into the E.coli high expression vector pMON5634 to create pMON5449. The endpoint atthe Bal31 created deletion was determined by DNA sequence analysis.

Construction of pMON5448 (XmnI, C-terminal Deletion of 16 bp)

pMON5436 was digested with XmnI and ligated with the syntheticterminator linker. The ligation was then digested with NcoI and BglIIand the 1.92 kb NcoI/BglII fragment containing the truncated gene wascloned into NcoI and BglII digested pMON9759 to replace the full-lengthgene and create pMON5446. The NcoI/HindIII fragment from pMON5446 wascloned into E, coli high expression vector pMON5634 to create pMON5448.

Construction of pMON5450 (NcoI fill-ends, Removal of First ATG fromToxin ORF

pMON5436 was digested with NcoI, the ends filled using Klenow fragmentDNA polymerase, ligated and transformed into JM101 to create pMON5450.This plasmid expresses only band 3 protein.

Construction of pMON5452 (N-terminal, Deletion of 224 bp)

The B.t. gene contains two StyI sites (227 and 1587) and a third sitewas added by the mutagenesis to create a NcoI site in pMON9759. Thefollowing experiments were performed to delete 5' B.t.t. DNA to basepair 227. pMON5434 (Hpal deletion derivative described above) wasdigested with StyI3 the ends filled with Klenow DNA polymerase, ligated,and transformed into JM101 to isolate pMON5444. This manipulationdestroys both the NcoI and StyI cleavage sites. This manipulationcreates an in frame fusion with the first methionine (amino acid 1) andleucine (amino acid 77). The C-terminus of the gene was added by cloningthe 1.9 kb NdeI/KpnI fragment from pMON9759 into pMON5444 to createpMON5452.

Construction of pMON5456 (Band 3 Mutant, N-terminal Deletion of 140 bp)

A NcoI site was introduced into pMON5420 at the ATG for band 3 by sitedirected mutagenesis as described above using the primer:

Mutagenesis Primer--BTTLOOP CGTATTATTATCTGCATCCATGGTTCTTCCTCCCT

to create pMON5455. The mutagenesis also deleted the upstream sequencewhich encodes the N-terminal 48 amino acids of band 1. The NcoI/HindIIIfragment from pMON5455 was cloned into the E. coli high expressionvector pMON5634 to create pMON5456. This plasmid expresses only band 3.The generation of the NcoI site changes the second amino acid fromthreonine to aspartic acid.

Construction of pMON5460 (Mutant Band 1 Gene with MET48 Changed to ILE)

The codon for methionine at position 48 in pMON9759 was changed to acodon for isoleucine by site directed mutagenesis as described aboveusing the primer:

Mutagenesis Primer--BTTMET ATTATTATCTGCAGTTATTCTTAAAAACTCTTTAT

to create pMON5458. The NcoI/HindIII fragment of pMON5458 was clonedinto the E. coli high expression vector pMON5634 to create pMON5460. Byremoving the ATG codon which initiates translation of band 3 protein,pMON5460 produces only band 1 protein with an isoleucine residue atposition 48.

Construction of pMON5467 (Band 5 Mutant, N-terminal Deletion of 293 bp)

A NcoI site was introduced into pMON5420 to create a N-terminal deletionof ninety-eight amino acids by site directed mutagenesis using theprimer:

Mutagenesis Primer TCACTTGGCCAAATTGCCATGGTATTTAAAAAGTTTGT

to create pMON5466. A methionine and alanine were also inserted by themutagenesis. The NcoI/HindIII fragment from pMON5466 was cloned into theE. coli high expression vector pMON5634 to create pMON5467.

Insect Toxicity Results

C-Terminal Truncations

Coleopteran-toxin activity was determined using newly hatched Coloradopotato beetles in a tomato leaf feeding assay as previously described.The mutant B.t.t. genes used for analysis of the C-terminus are shown inFIGS. 8 and 10. pMON5438 contains 490 amino acids of B.t.t. toxinprotein plus 3 amino acids encoded by the linker used in the vectorconstruction. The truncated protein was produced at high levels in E.coli, but had no activity against Colorado potato beetle. pMON5441produces a protein which contains 536 amino acids of the B.t.t. toxin.The truncated protein was produced at high levels in E. coli but had noactivity against Colorado potato beetle. pMON5449 contains 582 aminoacids of the B.t.t. protein plus two amino acids encoded by the linkerused in the vector construction. The truncated protein was produced athigh levels in E. coli, but had no activity against Colorado potatobeetle. pMON5448 contains 640 amino acids of the B.t.t. protein plus 2amino acids encoded by the linker used in the vector construction. Thetruncated protein was produced at high levels by E. coli, but theprotein had no activity against Colorado potato beetle. These resultssuggest that the C-terminus of the B.t.t. toxin protein is required fortoxicity to Colorado potato beetle. A deletion of only 4 amino(pMON5448) acids resulted in a complete loss of activity. These resultsare directly contrary to the reported literature with respect toLepidopteran-type B.t. toxins.

Results for N-Terminal Mutations and Deletions

The other mutant B.t.t. genes used for analysis of the N-terminus areshown in FIGS. 9 and 10. Analysis of protein produced by pMON5450revealed that band 3 production in E. coli was due to translationinitiation at MET48 rather than a product of protease cleavage. Toxicitystudies also showed that band 3 was toxic. pMON5456 produces a proteinwhich begins at amino acid 48 with amino acid 49 changed from threonineto aspartic acid. This protein was produced at high levels in E. coliand was toxic to Colorado potato beetle. pMON5452 produces a proteinwhich begins at amino acid 77. This protein was expressed in E. coli,and it had activity against Colorado potato beetle. pMON5467 produces aprotein which begins at amino acid 99 and has two amino acids added tothe N-terminus (methionine and alanine). This protein was produced in E.coli and exhibited no detectable activity against Colorado potatobeetle, however, the level of expression for this deletion variant wassignificantly lower than other variants. These results suggest that theN-terminus of the B.t.t. toxin protein can tolerate deletions. Adeletion of 76 amino acids exhibitied toxicity. A deletion of 99 aminoacids did, however, result in a loss of toxicity. pMON5460 contains amutation which changed methionine at position 48 to isoleucine toprevent production of band 3. The toxicity of band 1 produced bypMON5460 was equal to the toxicity of band 3 produced by pMON5456.

Construction of Plant Transformation Vectors

The B.t. var. tenebrionis toxin gene contained in pMON5420 was modifiedfor incorporation into plant expression vectors. A BglII site wasintroduced just upstream of the ATG codon which specifies the initiationof translation of the full-length B.t.t. toxin protein (referred to asband 1) using the site specific mutagenesis protocol of Kunkel (1985) aspreviously described. The sequence of the B.t.t. toxin gene in theregion of the initiator ATG is:

ATGATAAGAAAGGGAGGAAGAAAAATGAATCCGAACAATCGAAGTGAACATGATACAATAMetAsnProAsnAsnArgSerGluHisAspThrIle

The primer for this mutagenesis (bttbgl) was 27 nucleotides in lengthand has the sequence:

CGGATTCATT TTAGATCTTC CTCCCTT

Following mutagenesis a plasmid containing the new BglII site wasidentified by digestion with BglII and the change was verified by DNAsequence analysis. The resulting plasmid containing the B.t.t. toxingene with the new BglII site was designated pMON9758 (FIG. 11).

The B.t.t. toxin gene in pMON9758 was inserted into the expressioncassette vector pMON316 (Sanders et al., 1987). pMON316 contains theCaMV35S promoter and the 3' end from the nopaline synthase (NOS) genewith a BglII site for gene insertion between these two elements. PlasmidpMON9758 was digested with BglII and a fragment of approximately 2 kbwas isolated. This fragment extends from the BglII site just upstream ofthe ATG codon to a BglII site found approximately 350 bp downstream ofthe termination codon for the B.t.t. toxin gene. Thus, this fragmentcontains the complete coding sequence of the B.t.t. gene and also about350 bp of noncoding sequence 3' to the termination codon. This BglIIfragment was ligated with BglII digested pMON316. Followingtransformation into E. coli, a colony was identified in which the B.t.t.toxin gene was inserted into pMON316 such that the 5' end of the toxingene was adjacent to the CaMV35S promoter. This plasmid was designatedpMON9753. A plasmid containing the B.t.t. toxin gene in the oppositeorientation in pMON316 was isolated and designated pMON9754 (FIG. 11).

Both pMON9753 and pMON9754 were introduced by a triparental matingprocedure into the Agrobacterium tumefaciens strain ASE which contains adisarmed Ti plasmid. Cointegrates between pMON9753 or pMON9754 and thedisarmed Ti plasmid were identified as described by Fraley et al.(1985), and their structures confirmed by Southern analysis of totalAgrobacterium DNA.

Additional plant expression vectors containing the B.t.t. toxin genehave also been constructed (see FIGS. 12 and 13). In these vectors theB.t.t. toxin gene has been inserted into the plant expression vectorpMON893 (FIG. 14). Referring to FIG. 14, the expression cassette pMON893consists of the enhanced CaMV35S promoter and the 3' end includingpolyadenylation signals from a soybean gene encoding the alpha-primesubunit of beta-conglycinin (referred to below as the "7S gene").Between these two elements is a multi-linker containing multiplerestriction sites for the insertion of genes.

The enhanced CaMV35S promoter was constructed as follows. A fragment ofthe CaMV35S promoter extending between position -343 and +9 waspreviously constructed in pUC13 by Odell et al. (1985). This segmentcontains a region identified by Odell et al. (1985) as being necessaryfor maximal expression of the CaMV35S promoter. It was excised as aClaI-HindIII fragment, made blunt ended with DNA polymerase I (Klenowfragment) and inserted into the HincII site of pUC18. The upstreamregion of the 35S promoter was excised from this plasmid as aHindIII-EcoRV fragment (extending from -343 to -90) and inserted intothe same plasmid between the HindIII and PstI sites. The enhancedCaMV35S promoter thus contains a duplication of sequences between -343and -90 (see FIG. 18).

The 3' end of the 7S gene is derived from the 7S gene contained on theclone designated 17.1 (Schuler et al., 1982). This 3' end fragment,which includes the polyadenylation signals, extends from an AvaII sitelocated about 30 bp upstream of the termination codon for thebeta-conglycinin gene in clone 17.1 to an EcoRI site located about 450bp downstream of this termination codon.

The remainder of pMON893 contains a segment of pBR322 which provides anorigin of replication in E. coli and a region for homologousrecombination with the disarmed T-DNA in Agrobacterium strain ACO(described below); the oriV region from the broad host range plasmidRK2; the streptomycin resistance/spectinomycin resistance gene from Tn7;and a chimeric NPTII gene, containing the CaMV35S promoter and thenopaline synthase (NOS) 3' end, which provides kanamycin resistance intransformed plant cells.

pMON9753 contained approximately 400 bp of 3' noncoding sequence beyondthe termination codon. Since this region is not necessary for toxinproduction it was removed from the B.t.t. toxin gene segments insertedin pMON893. In order to create a B.t.t. toxin gene containing no 3'flanking sequence, a BglII site was introduced just after thetermination codon by the method of Kunkel (1985). The sequence of theB.t.t. toxin gene around the termination codon is: p1GTTTATATAGACAAAATTGAATTTATTCCAGTGAATTAAATTAACTAGAAAGTAAAGAAGVaLTyrIleAspLysIleGluPheIleProValAsnEnd

Mutagenesis was performed with a primer (bttcterm) of sequence:

CTTTCTAGTT AAAGATCTTT AATTCACTG

Mutagenesis of the B.t.t. toxin gene was performed in pMON9758. Aplasmid which contains the new BglII site was designated pMON9787 (FIG.12). Because pMON9787 contains a BglII site just upstream of the ATGinitiation codon, the full coding sequence for the B.t.t. toxin genewith essentially no 5' or 3' flanking sequence is contained on a BglIIfragment of about 1940 bp.

This 1940 bp fragment was isolated from pMON9787 and ligated with BglIIdigested pMON893. A plasmid in which the 5' end of the B.t.t. toxin genewas adjacent to the enhanced CaMV35S promoter was identified anddesignated pMON9791 (FIG. 12).

A variant of the full length B.t.t. toxin is produced in E. coli from asecond methionine initiator codon. This protein, designated "band 3",has been found to be as toxic to Colorado potato beetle as the fulllength toxin ("band 1"). It is possible that, as was the case for theB.t.k.. gene, truncated forms of the B.t.t. gene might be more easilyexpressed in plant cells. Therefore, a modified B.t.t. toxin gene wasconstructed in which the region upstream of the band 3 ATG codon hasbeen removed. In order to remove this sequence, a BglII site wasinserted just upstream of the band 3 ATG by the method of Kunkel (1985).The sequence surrounding the band 3 ATG is:

CCAAATCCAACACTAAGGATTTAAATTATAAAGAGTTTTTAAGAATGACTGCAGATAATProAsnProThrLeuGluAspLeuAsnTyrLysGluPheLeuArgMetThrAlaAspAsn

Mutagenesis was performed with primer (bttnterm) of sequence:

ATCTGCAGTC ATTGTAGATC TCTCTTTATA ATTT

Mutagenesis with this primer was performed on the B.t.t. toxin genecontained in pMON5420. A plasmid containing the new BglII site wasdesignated pMON9788. A truncated B.t.t. toxin gene beginning at thisband 3 BglII site and extending to the BglII site just distal to thetermination codon found in pMON9787 was constructed in pMON893 asfollows. pMON9788 (FIG. 13) was digested with BglII and XbaI and afragment of about 1250 bp was isolated. This fragment extends from theband 3 ATG to a unique XbaI site in the middle of the B.t.t. toxin gene.pMON9787 was also digested with BglII and XbaI, and a fragment of about550 bp was isolated. This fragment extends from the unique XbaI site inthe middle of the toxin gene to the BglII site just distal to thetermination codon. These two fragments were mixed and ligated with BglIIdigested pMON893. A plasmid was identified in which the 5' end to thetoxin gene was adjacent to the enhanced CaMV35S promoter and designatedpMON9792. pMON9792 contains a N-terminal truncated derivative of theB.t.t. toxin gene (FIG. 13) which encodes only band 3.

Both pMON9791 and pMON9792 were introduced into A. tumefaciens strainACO which contains a disarmed Ti plasmid. Cointegrates have beenselected and have been used in the transformation of tomato and potato.

ACO is a disarmed strain similar to pTiB6SE described by Fraley et al.(1985). For construction of ACO the starting Agrobacterium strain wasthe strain A208 which contains a nopaline-type Ti plasmid. The Tiplasmid was disarmed in a manner similar to that described by Fraley etal. (1985) so that essentially all of the native T-DNA was removedexcept for the left border and a few hundred base pairs of T-DNA insidethe left border. The remainder of the T-DNA extending to a point justbeyond the right border was replaced with a novel piece of DNA including(from left to right) a segment of pBR322, the oriV region from plasmidRK2, and the kanamycin resistance gene from Tn601. The pBR322 and oriVsegments are similar to the segments in pMON893 and provide a region ofhomology for cointegrate formation. The structure of the ACO Ti plasmidis shown in FIG. 17.

Chimeric B.t.t. Toxin Gene Using a Mas Promoter

The MAS promoter was isolated from pTiA6 as a 1.5 kb EcoRI-Clalfragment. This DNA fragment extends from the ClaI site at nucleotide20,138 to the EcoRI site at 21,631 in the sequence of Barker et al.(1983). Referring to FIG. 15, the EcoRI-ClaI fragment was ligated withthe binary vector pMON505 (Horsch et al. 1986) which had been previouslydigested with EcoRI and ClaI. The resulting plasmid was designatedpMON706. A fragment containing the NOS 31' end was inserted downstreamof the MAS promoter to obtain a MAS-NOS 3' expression cassette vector.The NOS 3' fragment was excised from pMON530 as a 300 bp BglII-BamH|fragment and inserted into BglII-digested pMON706. The resulting plasmidwas designated pMON707.

Plasmid pMON530 was constructed by cleavage of pMON200 with NdeI toremove a 900 bp NdeI fragment to create pMON503. Plasmid pMON503 wascleaved with HindIII and SmaI and mixed with plasmid pTJS75(Schmidhauser and Helinski, 1985) that had also been cleaved withHindIII and SmaI. A plasmid that contained the 3.8 kb HindIII-SmaIfragment of pTJS75 joined to the 8 kb HindIII-SmaI fragment of pMON503was isolated and designated pMON505. Next the CaMV35S-NOS3' cassette wastransferred to pMON505 by cleavage of pMON316 with StuI and HindIII andisolation of the 2.5 kb StuI-HindIII fragment containing theNOS-NPTII'-NOS marker and the CaMV35S-NOS3' cassette. This was added topMON505 DNA cleaved with StuI and HindIII. Following ligation andtransformation a plasmid carrying the CaMV35S-NOS3' cassette in pMON505was isolated and designated pMON530.

Since some binary vectors have greatly reduced frequencies oftransformation in tomato as compared to co-integrating vectors,(McCormick et al., 1986), the MAS-NOS 3' cassette was moved from pMON707into the co-integrating vector pMON200 (Fraley et al., 1985). PlasmidpMON200 was digested with StuI and HindIII and a 7.7 kb fragmentisolated by agarose gel electrophoresis. Plasmid pMON707 was similarlydigested with StuI and HindIII and a 3.5 kb StuI-HindIII fragmentcontaining the MAS-NOS 3' cassette was isolated by agarose gelelectrophoresis and recovery on a DEAE membranes with subsequent elutionwith 1M NaCl. These two DNA fragments were ligated and the resultingplasmid was designated pMON9741 (FIG. 15). This plasmid contains theMAS-NOS 3' cassette in the pMON200 co-integrating background.

Chimeric B.t.t. toxin genes driven by the MAS promoter are prepared bydigesting either pMON9791 or pMON9792 with BglII, recovering the toxinencoding fragment and moving this fragment into pMON9741 following theteachings provided herein.

These intermediate vectors may be used to transform plants to exhibittoxicity to Coleopteran insects susceptible to the B.t.t. toxin protein.

Coleopteran-Type Toxin Gene Expression in Plants

Tomato Plant Transformation

The A. tumefaciens strains pMON9753-ASE and pMON9754-ASE were used totransform tomato leaf discs by the method of McCormick et al. (1986).Transformed tomato plants were recovered as described and assayed forkanamycin resistance.

Insect Toxicity of Transgenic Tomato Plants

Tomato plants transformed, with the B.t.t. toxin gene contained inpMON9753 were assayed for expression of the toxin gene by bioassay withColorado potato beetle (Leptinotarsa decemlineata) insects. Leafcuttings from plants to be assayed were placed in petri dishescontaining water saturated filter paper. Ten or twenty newly hatchedpotato beetle insects were added to the leaf cuttings and allowed tofeed on the leaves. After four days the insects were scored formortality. In addition, insects were examined for evidence of slowedgrowth rate (stunting), and the leaf tissue remaining was examined todetermine relative feeding damage.

In each experiment many non-transformed plants were included ascontrols. Between 50 and 100 non-transformed plants have now beenassayed as controls. Of these control plants, more than 80% show nomortality to potato beetle; about 15% give 10% mortality; and, 5% orfewer show 20% mortality. Mortality of greater than 20% has not beenseen with a control plant.

Table VI below summarizes toxicity results obtained with severalpMON9753 transgenic tomato plants.

                  TABLE VI                                                        ______________________________________                                        Toxicity of Transgenic Tomato Plants Containing                               pMON9753 to Colorado Potato Beetle                                            Kanamycin.sup.1                                                                            Mortality of CPB (%)                                             Plant  Resistance                                                                              Assay #1   Assay #2 Assay #3                                 ______________________________________                                        794    R         30         20                                                810    n.d.      50         20       40                                       871    R         30         10 (stunted)                                      886    R         50         40                                                887    n.d.      20         30       30                                       1009   n.d.      50                                                           1044   R         20 (stunted)                                                 1046   R         40 (stunted)                                                                             20                                                ______________________________________                                         .sup.1 n.d. represents No Data                                           

As, shown in Table VI several plants have been recovered whichconsistently show higher levels of mortality of Colorado potato beetlethan non-transformed control plants. These results indicate that theB.t.t. toxin gene is being expressed at levels sufficient to kill asignificant number of the insects feeding on these plants.

Coleopteran Toxin Expression in Potato

Shoot tips of potato cultivar Kennebec are subcultured on mediacontaining MS major and minor salts, 0.17 g/l sodium dihydrogenphosphate, 0.4 mg/l thiamine-HC, 0.1 g/l inositol, 3% sucrose, 2.0 g/lGelrite (Kelco Co.) at pH 5.6. Cultures are grown for 4 weeks at 24° C.in a 16 hour photoperiod. Stem internodes are cut into approximately 8mm lengths and the cut surfaces are smeared with Agrobacterium strainpMON9753-ASE which has been streaked on an LB agar plate and grown for 2to 3 days. pMON9753-ASE which is described above contains the chimericB.t.t. toxin gene driven by the CaMV35S promoter. Alternatively,Agrobacterium strains pMON9791-ACO or pMON9792-ACO containing chimericB.t.t. toxin genes are used. Stem sections are placed on 0.8%agar-solidified medium containing salts and organic addenda as in Jarretet al. (1980), 3% sucrose, 3 mg/l BA and 0.1 mg/l NAA at pH 5.6. After 4days the explants are transferred to medium of the same composition butwith carbenicillin at 500 mg/l and kanamycin as the selective agent fortransformed plant cells at 100 mg/l. Four weeks later the explants aretransferred again to medium of the same composition but with GA₃ at 0.3mg/l as the sole hormone. Callus which developed in the presence of 100mg/l kanamycin are shown to contain the NPTII enzyme when tested by adot blot assay indicating that the potato cells are transformed.Uninoculated control tissue is inhibited at this concentration ofkanamycin. Transformed potato tissue expresses the B.t.t. toxin gene.B.t.t. toxin mRNA may be detected by Northern analysis and B.t.t. toxinprotein may be detected by immunoassay such as Western blot analysis.However, in many cases the most sensitive assay for the presence ofB.t.t. toxin is the insect bioassay. Colorado potato beetle larvaefeeding on the transformed tissue suffer from the effects of the toxin.

This procedure for producing kanamycin resistant transformed potatocells has also been successfully used to regenerate shoots. Shoots whichare 1 to 2 cm in length are removed from the explants and placed on theshoot tip maintenance medium described above where the shoots readilyroot.

Plants generated in this fashion are tested for transformation byassaying for expression of the NPTII enzyme and by the ability of stemsegments to form callus on kanamycin containing medium. Transformedplants express the B.t.t. toxin gene. B.t.t. toxin mRNA may be detectedby Northern analysis and B.t.t. toxin protein may be detected byimmiunoassay such as Western blot analysis. Colorado potato beetlelarvae feeding on the transformed tissue suffer from the effects of thetoxin.

Coleopteran Toxin Expression in Cotton

Cotton seeds are surface sterilized by first soaking them for 10 minutesin a detergent solution of water to which Sparkleen soap has been added,then by agitating them for 20 min. in a 30% Chlorox solution containing2 drops of Tween 20 per 400 mls before rinsing them twice with steriledistilled water. The seeds are then soaked in 0.4% benolate for 10 min.The benolate is poured off prior to placing the seeds aspetically ontoagar solidified half strength MS salts. Seeds are germinated for 3-10days in the dark at 32° C. The cotyledons and hypocotyls are thenremoved aspetically and segmented. The segments are placed onto 1) agarsolidified MS medium containing 3% glucose, 2 mg/l napthalene aceticacid (NAA), and 1 mg/l kinetin (Medium MSS) or 2) Gelrite solidified MSmedium containing 3% glucose, B5 vitamins, 100 mg/l inositol, 0.75 mg/lMgC1₂, 0.1 mg/l dichlorophenoxy acetic acid (2,4-D) and 0.1 or 0.5 mg/lkinetin (Medium MST). Callus is maintained in a 16/8 photoperiod at 28°C. on either of these media until embryogenesis is initiated. Subcultureof the embryogenic callus is made onto the same medium as for initiationbut containing 3% sucrose instead of glucose. Somatic embryos aregerminated by moving them onto Gelrite solidified Stewart's mediumwithout plant growth regulators but containing 0.75 g/l MgC1₂.Germinated embryos are moved to soil in a growth chamber where theycontinue to grow. Plants are then moved to the greenhouse in order toset seed and flower.

Transformation of cotton tissues and production of transformed callusand plants is accomplished as follows. Aseptic seedlings are prepared asfor plant regeneration. Hypocotyl and cotyledon segments are inoculatedwith liquid overnight Agrobacterium cultures or with Agrobacterium grownon nutrient plates. The explants are co-cultured for 2-3 days on MSS orMST medium containing 1/10 the concentration of MS salts. Explants areblotted on filter paper to remove excess bacteria and plated on MSS orMST medium containing 500 mg/l carbenicillin amd 30-100 mg/l kanamycin.Callus which is transformed will grow on this medium and produceembryos. The embryos are grown into plants as stated for regeneration.The plants are tested for transformation by assay for expression ofNPTII.

When the Agrobacterium strain used for transformation contains achimeric B.t.t. toxin gene such as pMON9753, pMON9791 or pMON9792, theB.t.t. toxin gene is expressed in the transformed callus, embryosderived from this callus, and in the transformed plants derived from theembryos. For all of these cases, expression of the B.t.t. toxin mRNA maybe detected by Northern analysis, and expression of the B.t.t. toxinprotein may be detected by immunoassay such as Western blot analysis.Insect bioassay may be the most sensitive measure for the presence oftoxin protein.

Insect toxicity of the callus, embryos or plants is assayed by bioassaywith boll weevil larvae (Anthonomous grandis). Boll weevil larvaefeeding on transformed cotton cells or plants expressing the B.t.t.toxin gene suffer from the effects of the toxin.

Coleopteran Toxin Gene Expression in Maize

The following description outlines the preparation of protoplasts frommaize, the introduction of chimeric B.t.t. toxin genes into theprotoplast by electroporation, and the recovery of stably transformed,kanamycin resistant maize cells expressing chimeric B.t.t. toxin genes.

Preparation of Maize Protoplasts

Protoplasts are prepared from a Black Mexican Sweet (BMS) maizesuspension line, BMSI (ATCC 54022) as described by Fromm et al. (1985and 1986). BMSI suspension cells are grown in BMS medium which containsMS salts, 20 g/l. sucrose, 2 mg/l (2,4-dichlorophenoxy) acetic acid, 200mg/l inositol, 130 mg/l asparageine, 1.3 mg/l niacin, 0.25 mg/lthiamine, 0.25 mg/l pyridoxine, 0.25 mg/l calcium pantothenate, pH 5.8Forty ml cultures in 125 ml erlenmeyer flasks are shaken at 150 rpm at26° C. The culture is diluted with an equal volume of fresh medium every3 days. Protoplasts are isolated from actively growing cells 1 to 2 daysafter adding fresh medium. For protoplast isolation cells are pelletedat 200×g in a swinging bucket table top centrifuge. The supernatant issaved as conditioned medium for culturing the protoplasts. Six ml ofpacked cells are resuspended in 40 ml of 0.2 M mannitol/50 mM CaCI₂ /10mM sodium acetate which contains 1% cellulase, 0.5% hemicellulase and0.02% pectinase. After incubation for 2 hours at 26° C., protoplasts areseparated by filtration through a 60 μm nylon mesh screen, centriguredat 200×g, and washed once in the same solution without enzymes.

Transformation of Maize Protoplasts with B.t.t. Toxin Gene DNA VectorsUsing an Electroporation Technique

Protoplasts are prepared for electroporation by washing in a solutioncontaining 2 mM potassium phosphate pH 7.1, 4 mM calcium chloride, 140mM sodium chloride and 0.2M mannitol. After washing, the protoplasts areresuspended in the same solution at a concentration of 4×10⁶ protoplastsper ml. One-half ml of the protoplast containing solution is mixed with0.5 ml of the same solution containing 50 micrograms of supercoiledplasmid vector DNA and placed in a 1 ml an electroporation cuvette.Electroporation is carried out as described by Fromm et al. (1986). Asdescribed, an electrical pulse is delivered from a 122 or 245 microFaradcapacitor charged to 200 V. After 10 min. at 4° C. and 10 min. at roomtemperature protoplasts are diluted with 8 ml of medium containing MSsalts 0.3M mannitol, 2% sucrose, 2 mg/l 2,4-D, 20% conditioned BMSmedium (see above) and 0.1% low melting agarose. After 2 weeks in thedark at 26° C., medium without mannitol and containing kanamycin isadded to give a final kanamycin concentration of 100 mg/l liquid. Afteran additional 2 weeks, microcalli are removed from the liquid and placedon a membrane filter disk above agarose solidified medium containing 100mg/l kanamycin. Kanamycin resistant calli composed of transformed maizecells appear after about 1-2 weeks.

Expression of B.t.tToxin Genes in Maize Cells

As described by Fromm et al. (1986), transformed maize cells can beselected by growth in kanamycin containing medium followingelectroporation with DNA vectors containing chimeric kanamycinresistance genes composed of the CaMV35S promoter, the NPTII codingregion and the NOS 3' end. pMON9791 and pMON9792 contain such chimericNPTII genes and also contain chimeric B.t.t. toxin genes. As describedabove, maize protoplasts are transformed by electroporation with DNAvectors where the DNA vectors are pMON9791 or pMON9792. Followingselection for kanamycin resistance, the transformed maize cells areassayed for expression of the B.t.t. toxin gene. Assays are performedfor B.t.t. mRNA by Northern blot analysis and for B.t.t. toxin proteinby immunoassay such as Western blot analysis.

Assays for insect toxicity are performed by feeding transformed maizecalli to Southern corn rootworm larvae (Diabrotica undecimpunctatahowardi). Alternatively, a protein extract containing the B.t.t. toxinprotein is prepared from transformed maize cells and this extract isincorporated into an appropriate insect diet which is fed to theSouthern corn rootworm larvae. Rootworm larvae feeding on transformedcalli or protein extracts of such calli suffer from the effects of thetoxin.

The above examples are provided to better elucidate the practice of thepresent invention and are not intended, in any way, to limit the scopeof the present invention. Those skilled in the art will recognize thatmodifications may be made without deviating from the spirit and scope ofthe invention as described.

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M13 Cloning and Sequencing Handbook, Amersham Corporation Cat. #N4502.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2615 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 205..2139                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GAGCGACTATTATAATCATACATATTTTCTATTGGAATGATTAAGATTCCAATAGAATAG60                TGTATAAATTATTTATCTTGAAAGGAGGGATGCCTAAAAACGAAGAACATTAAAAACATA120               TATTTGCACCGTCTAATGGATTTATGAAAAATCATTTTATCAGTTTGAAAATTATGTATT180               ATGATAAGAAAGGGAGGAAGAAAAATGAATCCGAACAATCGAAGTGAACAT231                        MetAsnProAsnAsnArgSerGluHis                                                   15                                                                            GATACAATAAAAACTACTGAAAATAATGAGGTGCCAACTAACCATGTT279                           AspThrIleLysThrThrGluAsnAsnGluValProThrAsnHisVal                              10152025                                                                      CAATATCCTTTAGCGGAAACTCCAAATCCAACACTAGAAGATTTAAAT327                           GlnTyrProLeuAlaGluThrProAsnProThrLeuGluAspLeuAsn                              303540                                                                        TATAAAGAGTTTTTAAGAATGACTGCAGATAATAATACGGAAGCACTA375                           TyrLysGluPheLeuArgMetThrAlaAspAsnAsnThrGluAlaLeu                              455055                                                                        GATAGCTCTACAACAAAAGATGTCATTCAAAAAGGCATTTCCGTAGTA423                           AspSerSerThrThrLysAspValIleGlnLysGlyIleSerValVal                              606570                                                                        GGTGATCTCCTAGGCGTAGTAGGTTTCCCGTTTGGTGGAGCGCTTGTT471                           GlyAspLeuLeuGlyValValGlyPheProPheGlyGlyAlaLeuVal                              758085                                                                        TCGTTTTATACAAACTTTTTAAATACTATTTGGCCAAGTGAAGACCCG519                           SerPheTyrThrAsnPheLeuAsnThrIleTrpProSerGluAspPro                              9095100105                                                                    TGGAAGGCTTTTATGGAACAAGTAGAAGCATTGATGGATCAGAAAATA567                           TrpLysAlaPheMetGluGlnValGluAlaLeuMetAspGlnLysIle                              110115120                                                                     GCTGATTATGCAAAAAATAAAGCTCTTGCAGAGTTACAGGGCCTTCAA615                           AlaAspTyrAlaLysAsnLysAlaLeuAlaGluLeuGlnGlyLeuGln                              125130135                                                                     AATAATGTCGAAGATTATGTGAGTGCATTGAGTTCATGGCAAAAAAAT663                           AsnAsnValGluAspTyrValSerAlaLeuSerSerTrpGlnLysAsn                              140145150                                                                     CCTGTGAGTTCACGAAATCCACATAGCCAGGGGCGGATAAGAGAGCTG711                           ProValSerSerArgAsnProHisSerGlnGlyArgIleArgGluLeu                              155160165                                                                     TTTTCTCAAGCAGAAAGTCATTTTCGTAATTCAATGCCTTCGTTTGCA759                           PheSerGlnAlaGluSerHisPheArgAsnSerMetProSerPheAla                              170175180185                                                                  ATTTCTGGATACGAGGTTCTATTTCTAACAACATATGCACAAGCTGCC807                           IleSerGlyTyrGluValLeuPheLeuThrThrTyrAlaGlnAlaAla                              190195200                                                                     AACACACATTTATTTTTACTAAAAGACGCTCAAATTTATGGAGAAGAA855                           AsnThrHisLeuPheLeuLeuLysAspAlaGlnIleTyrGlyGluGlu                              205210215                                                                     TGGGGATACGAAAAAGAAGATATTGCTGAATTTTATAAAAGACAACTA903                           TrpGlyTyrGluLysGluAspIleAlaGluPheTyrLysArgGlnLeu                              220225230                                                                     AAACTTACGCAAGAATATACTGACCATTGTGTCAAATGGTATAATGTT951                           LysLeuThrGlnGluTyrThrAspHisCysValLysTrpTyrAsnVal                              235240245                                                                     GGATTAGATAAATTAAGAGGTTCATCTTATGAATCTTGGGTAAACTTT999                           GlyLeuAspLysLeuArgGlySerSerTyrGluSerTrpValAsnPhe                              250255260265                                                                  AACCGTTATCGCAGAGAGATGACATTAACAGTATTAGATTTAATTGCA1047                          AsnArgTyrArgArgGluMetThrLeuThrValLeuAspLeuIleAla                              270275280                                                                     CTATTTCCATTGTATGATGTTCGGCTATACCCAAAAGAAGTTAAAACC1095                          LeuPheProLeuTyrAspValArgLeuTyrProLysGluValLysThr                              285290295                                                                     GAATTAACAAGAGACGTTTTAACAGATCCAATTGTCGGAGTCAACAAC1143                          GluLeuThrArgAspValLeuThrAspProIleValGlyValAsnAsn                              300305310                                                                     CTTAGGGGCTATGGAACAACCTTCTCTAATATAGAAAATTATATTCGA1191                          LeuArgGlyTyrGlyThrThrPheSerAsnIleGluAsnTyrIleArg                              315320325                                                                     AAACCACATCTATTTGACTATCTGCATAGAATTCAATTTCACACGCGG1239                          LysProHisLeuPheAspTyrLeuHisArgIleGlnPheHisThrArg                              330335340345                                                                  TTCCAACCAGGATATTATGGAAATGACTCTTTCAATTATTGGTCCGGT1287                          PheGlnProGlyTyrTyrGlyAsnAspSerPheAsnTyrTrpSerGly                              350355360                                                                     AATTATGTTTCAACTAGACCAAGCATAGGATCAAATGATATAATCACA1335                          AsnTyrValSerThrArgProSerIleGlySerAsnAspIleIleThr                              365370375                                                                     TCTCCATTCTATGGAAATAAATCCAGTGAACCTGTACAAAATTTAGAA1383                          SerProPheTyrGlyAsnLysSerSerGluProValGlnAsnLeuGlu                              380385390                                                                     TTTAATGGAGAAAAAGTCTATAGAGCCGTAGCAAATACAAATCTTGCG1431                          PheAsnGlyGluLysValTyrArgAlaValAlaAsnThrAsnLeuAla                              395400405                                                                     GTCTGGCCGTCCGCTGTATATTCAGGTGTTACAAAAGTGGAATTTAGC1479                          ValTrpProSerAlaValTyrSerGlyValThrLysValGluPheSer                              410415420425                                                                  CAATATAATGATCAAACAGATGAAGCAAGTACACAAACGTACGACTCA1527                          GlnTyrAsnAspGlnThrAspGluAlaSerThrGlnThrTyrAspSer                              430435440                                                                     AAAAGAAATGTTGGCGCGGTCAGCTGGGATTCTATCGATCAATTGCCT1575                          LysArgAsnValGlyAlaValSerTrpAspSerIleAspGlnLeuPro                              445450455                                                                     CCAGAAACAACAGATGAACCTCTAGAAAAGGGATATAGCCATCAACTC1623                          ProGluThrThrAspGluProLeuGluLysGlyTyrSerHisGlnLeu                              460465470                                                                     AATTATGTAATGTGCTTTTTAATGCAGGGTAGTAGAGGAACAATCCCA1671                          AsnTyrValMetCysPheLeuMetGlnGlySerArgGlyThrIlePro                              475480485                                                                     GTGTTAACTTGGACACATAAAAGTGTAGACTTTTTTAACATGATTGAT1719                          ValLeuThrTrpThrHisLysSerValAspPhePheAsnMetIleAsp                              490495500505                                                                  TCGAAAAAAATTACACAACTTCCGTTAGTAAAGGCATATAAGTTACAA1767                          SerLysLysIleThrGlnLeuProLeuValLysAlaTyrLysLeuGln                              510515520                                                                     TCTGGTGCTTCCGTTGTCGCAGGTCCTAGGTTTACAGGAGGAGATATC1815                          SerGlyAlaSerValValAlaGlyProArgPheThrGlyGlyAspIle                              525530535                                                                     ATTCAATGCACAGAAAATGGAAGTGCGGCAACTATTTACGTTACACCG1863                          IleGlnCysThrGluAsnGlySerAlaAlaThrIleTyrValThrPro                              540545550                                                                     GATGTGTCGTACTCTCAAAAATATCGAGCTAGAATTCATTATGCTTCT1911                          AspValSerTyrSerGlnLysTyrArgAlaArgIleHisTyrAlaSer                              555560565                                                                     ACATCTCAGATAACATTTACACTCAGTTTAGACGGGGCACCATTTAAT1959                          ThrSerGlnIleThrPheThrLeuSerLeuAspGlyAlaProPheAsn                              570575580585                                                                  CAATACTATTTCGATAAAACGATAAATAAAGGAGACACATTAACGTAT2007                          GlnTyrTyrPheAspLysThrIleAsnLysGlyAspThrLeuThrTyr                              590595600                                                                     AATTCATTTAATTTAGCAAGTTTCAGCACACCATTCGAATTATCAGGG2055                          AsnSerPheAsnLeuAlaSerPheSerThrProPheGluLeuSerGly                              605610615                                                                     AATAACTTACAAATAGGCGTCACAGGATTAAGTGCTGGAGATAAAGTT2103                          AsnAsnLeuGlnIleGlyValThrGlyLeuSerAlaGlyAspLysVal                              620625630                                                                     TATATAGACAAAATTGAATTTATTCCAGTGAATTAAATTAACTAGAAAGTAAA2156                     TyrIleAspLysIleGluPheIleProValAsn                                             635640645                                                                     GAAGTAGTGACCATCTATGATAGTAAGCAAAGGATAAAAAAATGAGTTCATAAAATGAAT2216              AACATAGTGTTCTTCAACTTTCGCTTTTTGAAGGTAGATGAAGAACACTATTTTTATTTT2276              CAAAATGAAGGAAGTTTTAAATATGTAATCATTTAAAGGGAACAATGAAAGTAGGAAATA2336              AGTCATTATCTATAACAAAATAACCATTTTTATATAGCCAGAAATGAATTATAATATTAA2396              TCTTTTCTAAATTGACGTTTTTCTAAACGTTCTATAGCTTCAAGACGCTTAGAATCATCA2456              ATATTTGTATACAGAGCTGTTGTTTCCATCGAGTTATGTCCCATTTGATTCGCTAATAGA2516              ACAAGATCTTTATTTTCGTTATAATGATTGGTTGCATAAGTATGGCGTAATTTATGAGGG2576              CTTTTCTTTTCATCCAAAAGCCAAGTGTATTTCTCTGTA2615                                   (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 644 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetAsnProAsnAsnArgSerGluHisAspThrIleLysThrThrGlu                              151015                                                                        AsnAsnGluValProThrAsnHisValGlnTyrProLeuAlaGluThr                              202530                                                                        ProAsnProThrLeuGluAspLeuAsnTyrLysGluPheLeuArgMet                              354045                                                                        ThrAlaAspAsnAsnThrGluAlaLeuAspSerSerThrThrLysAsp                              505560                                                                        ValIleGlnLysGlyIleSerValValGlyAspLeuLeuGlyValVal                              65707580                                                                      GlyPheProPheGlyGlyAlaLeuValSerPheTyrThrAsnPheLeu                              859095                                                                        AsnThrIleTrpProSerGluAspProTrpLysAlaPheMetGluGln                              100105110                                                                     ValGluAlaLeuMetAspGlnLysIleAlaAspTyrAlaLysAsnLys                              115120125                                                                     AlaLeuAlaGluLeuGlnGlyLeuGlnAsnAsnValGluAspTyrVal                              130135140                                                                     SerAlaLeuSerSerTrpGlnLysAsnProValSerSerArgAsnPro                              145150155160                                                                  HisSerGlnGlyArgIleArgGluLeuPheSerGlnAlaGluSerHis                              165170175                                                                     PheArgAsnSerMetProSerPheAlaIleSerGlyTyrGluValLeu                              180185190                                                                     PheLeuThrThrTyrAlaGlnAlaAlaAsnThrHisLeuPheLeuLeu                              195200205                                                                     LysAspAlaGlnIleTyrGlyGluGluTrpGlyTyrGluLysGluAsp                              210215220                                                                     IleAlaGluPheTyrLysArgGlnLeuLysLeuThrGlnGluTyrThr                              225230235240                                                                  AspHisCysValLysTrpTyrAsnValGlyLeuAspLysLeuArgGly                              245250255                                                                     SerSerTyrGluSerTrpValAsnPheAsnArgTyrArgArgGluMet                              260265270                                                                     ThrLeuThrValLeuAspLeuIleAlaLeuPheProLeuTyrAspVal                              275280285                                                                     ArgLeuTyrProLysGluValLysThrGluLeuThrArgAspValLeu                              290295300                                                                     ThrAspProIleValGlyValAsnAsnLeuArgGlyTyrGlyThrThr                              305310315320                                                                  PheSerAsnIleGluAsnTyrIleArgLysProHisLeuPheAspTyr                              325330335                                                                     LeuHisArgIleGlnPheHisThrArgPheGlnProGlyTyrTyrGly                              340345350                                                                     AsnAspSerPheAsnTyrTrpSerGlyAsnTyrValSerThrArgPro                              355360365                                                                     SerIleGlySerAsnAspIleIleThrSerProPheTyrGlyAsnLys                              370375380                                                                     SerSerGluProValGlnAsnLeuGluPheAsnGlyGluLysValTyr                              385390395400                                                                  ArgAlaValAlaAsnThrAsnLeuAlaValTrpProSerAlaValTyr                              405410415                                                                     SerGlyValThrLysValGluPheSerGlnTyrAsnAspGlnThrAsp                              420425430                                                                     GluAlaSerThrGlnThrTyrAspSerLysArgAsnValGlyAlaVal                              435440445                                                                     SerTrpAspSerIleAspGlnLeuProProGluThrThrAspGluPro                              450455460                                                                     LeuGluLysGlyTyrSerHisGlnLeuAsnTyrValMetCysPheLeu                              465470475480                                                                  MetGlnGlySerArgGlyThrIleProValLeuThrTrpThrHisLys                              485490495                                                                     SerValAspPhePheAsnMetIleAspSerLysLysIleThrGlnLeu                              500505510                                                                     ProLeuValLysAlaTyrLysLeuGlnSerGlyAlaSerValValAla                              515520525                                                                     GlyProArgPheThrGlyGlyAspIleIleGlnCysThrGluAsnGly                              530535540                                                                     SerAlaAlaThrIleTyrValThrProAspValSerTyrSerGlnLys                              545550555560                                                                  TyrArgAlaArgIleHisTyrAlaSerThrSerGlnIleThrPheThr                              565570575                                                                     LeuSerLeuAspGlyAlaProPheAsnGlnTyrTyrPheAspLysThr                              580585590                                                                     IleAsnLysGlyAspThrLeuThrTyrAsnSerPheAsnLeuAlaSer                              595600605                                                                     PheSerThrProPheGluLeuSerGlyAsnAsnLeuGlnIleGlyVal                              610615620                                                                     ThrGlyLeuSerAlaGlyAspLysValTyrIleAspLysIleGluPhe                              625630635640                                                                  IleProValAsn                                                                  __________________________________________________________________________

We claim:
 1. A method for producing a genetically transformed plantwhich exhibits toxicity toward Coleopteran insects which comprises thesteps of:(a) inserting into the genome of a plant cell a chimeric genewhich comprises in sequence:i) a promoter which functions in plants tocause the production of RNA; ii) a DNA sequence that causes theproduction of a RNA sequence encoding Coleopterantype toxin protein ofBacillus thuringiensis var. tenebrionis having the amino acid sequenceselected from the group consisting of from residues (1-644), residues(16-644), residues (48-644), residues (50-644), residues (58-644) andresidues (77-644) of said protein wherein the amino acid residues ofsaid protein are numbered as shown in FIG. 10; and iii) a 3'non-translated DNA sequence which functions in plant cells to cause theaddition of polyadenylate nucleotides to the 3' end of the RNA sequence;(b) obtaining transformed plant cells; and (c) regenerating from thetransformed plant cells genetically transformed plants exhibitingresistance to Coleopteran insects.
 2. The method of claim 1 in which thepromoter is selected from the group consisting of the CaMV35 S promoter,the MAS promoter and the ssRUBISCO promoter.
 3. The method of claim 2 inwhich the promoter is the CaMV35S promoter.
 4. The method of claim 2 inwhich the promoter is the MAS promoter.
 5. The method of claim 3 inwhich the 3' non-translated DNA sequence is from the soybean storageprotein gene.
 6. The method of claim 1 in which the plant is selectedfrom the group consisting of tomato, potato and cotton.
 7. The method ofclaim 5 which further comprises an enhancer sequence 5' from thepromoter.
 8. The method of claim 7 in which the promoter is the CaMV35Spromoter and the enhancer sequence has the nucleotide sequence of fromresidues 47-279 as shown in FIG.
 18. 9. The method of claim 1 in whichsaid DNA sequence encodes the toxin protein of Bacillus thuringiensisvar. tenebrionis having the amino acid sequence from residues (1-644) ofsaid protein wherein the amino acid residues of said protein arenumbered as shown in FIG.
 10. 10. The method of claim 1 in which saidDNA sequence encodes the toxin protein of Bacillus thuringiensis var.tenebrionis having the amino acid sequence from residues (16-644) ofsaid protein wherein the amino acid residues of said protein arenumbered as shown in FIG.
 10. 11. A method for producing a geneticallytransformed plant which exhibits toxicity toward Coleopteran insectswhich comprises the steps of:(a) inserting into the genome of a plantcell a chimeric gene which comprises in sequence:i) a promoter whichfunctions in plants to cause the production of RNA; ii) a DNA sequencethat causes the production of a RNA sequence encoding Coleopterantypetoxin protein of Bacillus thuringiensis var. tenebrionis having theamino acid sequence from residues (48-644) of said protein wherein theamino acid residues of said protein are numbered as shown in FIG. 10;and iii) a 3' non-translated DNA sequence which functions in plant cellsto cause the addition of polyadenylate nucleotides to the 3' end of theRNA sequence; (b) obtaining transformed plant cells; and (c)regenerating from the transformed plant cells genetically transformedplants exhibiting resistance to Coleopteran insects.
 12. The method ofclaim 1 in which said DNA sequence encodes the toxin protein of Bacillusthuringiensis var. tenebrionis having the amino acid sequence fromresidues (50-644) of said protein wherein the amino acid residues ofsaid protein are numbered as shown in FIG.
 10. 13. The method of claim 1in which said DNA sequence encodes the toxin protein of Bacillusthuringiensis var. tenebrionis having the amino acid sequence fromresidues (58-644) of said protein wherein the amino acid residues ofsaid protein are numbered as shown in FIG.
 10. 14. The method of claim 1in which said DNA sequence encodes the toxin protein of Bacillusthuringiensis var. tenebrionis having the amino acid sequence fromresidues (77-644) of said protein wherein the amino acid residues ofsaid protein are numbered as shown in FIG. 10.